Induction of human embryonic stem cell derived cardiac pacemaker or chamber-type cardiomyocytes by manipulation of neuregulin signaling

ABSTRACT

The present invention is directed to methods of producing cardiomyocytes having a nodal/pacemaker phenotype and cardiomyocytes having an atrial/ventricular phenotype. Isolated populations of nodal/pacemaker and atrial/ventricular cardiomyocytes are also disclosed. Methods of treating a subject having cardiac arrhythmia and a subject in need of cardiac tissue repair using the isolated populations of nodal/pacemaker cardiomyocytes and atrial/ventricular cardiomyocytes, respectively, are also disclosed.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/110,793, filed Nov. 3, 2008, which is herebyincorporated by reference in its entirety.

This invention was made with government support under grant numbers K08HL080431 and R01 HL064387 awarded by the National Institutes of Healthand National Heart, Lung, and Blood Institute. The government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods of producing nodal/pacemakerand atrial/ventricular cardiomyocytes. The invention further relates tomethods of treating cardiac arrhythmias and improving cardiac tissuerepair and function using the nodal/pacemaker and atrial/ventricularcell populations, respectively.

BACKGROUND OF THE INVENTION Prospects for Cell-Based Cardiac Therapieswith Cardiomyocytes from Pluripotent Human Cells

The muscle lost after a myocardial infarction is replaced withnon-contractile scar tissue, often initiating heart failure. Whole-organcardiac transplantation is the only currently available clinical meansof replacing the lost muscle, but this option is limited by theinadequate supply of donor hearts. Given this, much attention hasrecently been directed at cell transplantation strategies as analternate means of ameliorating cardiac injury (Laflamme & Murry,“Regenerating the Heart,” Nat Biotechnol 23(7):845-856 (2005); Dimmeleret al., “Unchain My Heart: The Scientific Foundations of CardiacRepair,” J Clin Invest 115(3):572-583 (2005); Laflamme et al.,“Cell-Based Cardiac Repair: Pathophysiologic Mechanisms,” Annual RevPathol 2:307-39 (2007); Rubart et al., “Cardiac Regeneration:Repopulating the Heart,” Annu Rev Physiol 68:29-49 (2006)). A number ofcandidate cell types have been considered, including skeletal myoblasts(Menasche et al., “Autologous Skeletal Myoblast Transplantation forSevere Postinfarction Left Ventricular Dysfunction,” J Am Coll Cardiol41(7):1078-1083 (2003); Murry et al., “Skeletal Myoblast Transplantationfor Repair of Myocardial Necrosis,” J Clin Invest 98(11):2512-2523(1996); Taylor et al., “Regenerating Functional Myocardium: ImprovedPerformance After Skeletal Myoblast Transplantation,” Nat Med4(8):929-933 (1998)), bone marrow-derived hematopoietic stem cells(Orlic et al., “Bone Marrow Cells Regenerate Infarcted Myocardium,”Nature 410(6829):701-705 (2001)), mesenchymal stem cells (Shake et al.,“Mesenchymal Stem Cell Implantation in a Swine Myocardial Infarct Model:Engraftment and Functional Effects,” Ann Thorac Surg 73(6):1919-1926(2002); Toma et al., “Human Mesenchymal Stem Cells Differentiate to aCardiomyocyte Phenotype in the Adult Murine Heart,” Circulation105(1):93-98 (2002); Min et al., “Significant Improvement of HeartFunction By Co-transplantation of Human Mesenchymal Stem Cells and FetalCardiomyocytes in Postinfarcted Pigs,” Ann Thorac Surg 74(5):1568-1575(2002); Mangi et al., “Mesenchymal Stem Cells Modified With Akt PreventRemodeling and Restore Performance of Infarcted Hearts,” Nat Med9(9):1195-1201 (2003)), and intrinsic cardiac stem cells (Beltrami etal., “Adult Cardiac Stem Cells Are Multipotent and Support MyocardialRegeneration,” Cell 114(6):763-776 (2003); Oh et al., “CardiacProgenitor Cells From Adult Myocardium Homing, Differentiation, andFusion After Infarction,” Proc Natl Acad Sci USA 100(21):12313-12318(2003); Laugwitz et al., “Postnatal isl1+ Cardioblasts Enter FullyDifferentiated Cardiomyocyte Lineages,” Nature 433(7026):647-653 (2005);Smith et al., “Regenerative Potential of Cardiosphere-Derived CellsExpanded From Percutaneous Endomyocardial Biopsy Specimens,” Circulation115(7):896-908 (2007)), but pluripotent hESCs have a number ofparticularly attractive properties for such applications. First, incontrast to many adult stem cell types for which this capacity iscontroversial, hESCs have unquestioned cardiomyogenic potential (Xu etal., “Characterization and Enrichment of Cardiomyocytes Derived FromHuman Embryonic Stem Cells,” Circ Res 91(6):501-508 (2002); Mummery etal., “Differentiation of Human Embryonic Stem Cells to Cardiomyocytes:Role of Coculture With Visceral Endoderm-Like Cells,” Circulation107(21):2733-2740 (2003); Kehat et al., “Human Embryonic Stem Cells CanDifferentiate Into Myocytes With Structural and Functional Properties ofCardiomyocytes,” J Clin Invest 108(3):407-414 (2001)). Indeed, anefficient protocol that reliably generates large quantities of ˜90%cardiomyocytes from hESCs, has greatly facilitated work with these cells(Laflamme et al., “Cardiomyocytes Derived From Human Embryonic StemCells in Pro-Survival Factors Enhance Function of Infarcted Rat Hearts,”Nat Biotechnol 25(9):1015-1024 (2007)). Second, hESCs can be isolatedand maintained by well-established protocols, and they are tremendouslyscaleable. Undifferentiated hESCs retain their phenotype through as manyas one hundred population doublings, and, after differentiation,hESC-CMs exhibit robust proliferative capacity both in vitro (Xu et al.,“Characterization and Enrichment of Cardiomyocytes Derived From HumanEmbryonic Stem Cells,” Circ Res 91(6):501-508 (2002); McDevitt et al.,“Proliferation of Cardiomyocytes Derived From Human Embryonic Stem Cellsis Mediated Via the IGF/PI 3-kinase/Akt Signaling Pathway,” J Mol CellCardiol 39(6):865-873 (2005); Snir et al., “Assessment of theUltrastructural and Proliferative Properties of Human Embryonic StemCell-Derived Cardiomyocytes,” Am J Physiol Heart Circ Physiol285(6):H2355-2363 (2003)) and in vivo (Laflamme et al., “Formation ofHuman Myocardium in the Rat Heart From Human Embryonic Stem Cells,” Am JPathol 167(3):663-671 (2005)). Third, a number of recent reports havedemonstrated that hESC-CMs survive after transplantation into infarctedrodent hearts, form stable cardiac implants, and preserve contractilefunction (Laflamme et al., “Cardiomyocytes Derived From Human EmbryonicStem Cells in Pro-Survival Factors Enhance Function of Infarcted RatHearts,” Nat Biotechnol 25(9):1015-1024 (2007); Caspi et al.,“Transplantation of Human Embryonic Stem Cell-Derived CardiomyocytesImproves Myocardial Performance in Infarcted Rat Hearts,” J Am CollCardiol 50(19):1884-1893 (2007); van Laake et al., “Human Embryonic StemCell-Derived Cardiomyocytes Survive and Mature in the Mouse Heart andTransiently Improve Function After Myocardial Infarction,” Stem CellResearch 1(1):9-24 (2007)). Finally, although the need to useimmunosuppression to overcome allorejection of hESC-derivatives hasoften been raised as a limitation of these cells, the recently reportedESC-like induced pluripotent stem cells (iPSCs) represent a potentialsolution to this problem (Takahashi et al., “Induction of PluripotentStem Cells From Adult Human Fibroblasts By Defined Factors,” Cell131(5):861-872 (2007); Park et al., “Reprogramming of Human SomaticCells to Pluripotency With Defined Factors,” Nature (2007); Yu et al.,“Induced Pluripotent Stem Cell Lines Derived From Human Somatic Cells,”Science 318(5858):1917-1920 (2007)). iPSCs are generated byreprogramming human adult fibroblasts and so may be used in anautologous fashion. iPSCs can be induced to differentiate intocardiomyocytes using the same techniques as those reported for hESCs(Takahashi et al., “Induction of Pluripotent Stem Cells From Adult HumanFibroblasts By Defined Factors,” Cell 131(5):861-872 (2007)).

Often overlooked is another potential application for cell-basedtherapies: the development of a “biological pacemaker”. An estimatedthree million people worldwide currently have an implanted artificialpacemaker, and up to 600,000 receive new ones each year (Wood et al.,“Cardiology Patient Pages. Cardiac Pacemakers From the Patient'sPerspective,” Circulation 105(18):2136-2138 (2002)). While these devicessuccessfully treat a broad range of electrophysiological abnormalities,they do have shortcomings including increased susceptibility toinfection, a finite battery life, significant patient discomfort, andlack of intrinsic responsiveness to neurohumoral signaling. Theselimitations have led to recent interest in gene- and/or cell-basedtherapies as an alternate strategy (Miake et al., “Biological PacemakerCreated By Gene Transfer,” Nature 419(6903):132-133 (2002); Qu et al.,“Expression and Function of a Biological Pacemaker in Canine Heart,”Circulation 107(8):1106-1109 (2003); Plotnikov et al., “BiologicalPacemaker Implanted in Canine Left Bundle Branch Provides VentricularEscape Rhythms That Have Physiologically Acceptable Rates,” Circulation109(4):506-512 (2004); Potapova et al., “Human Mesenchymal Stem Cells asa Gene Delivery System to Create Cardiac Pacemakers,” Circ Res94(7):952-959 (2004)).

Signaling Pathways Involved in Development of the Cardiac Pacemaking andConduction Systems

Elegant work in developmental model systems has implicated a variety ofsignaling molecules as important in the development of “specialized”cardiac subtypes, including neuregulin (NRG) (Hertig et al.,“Synergistic Roles of Neuregulin-1 and Insulin-Like Growth Factor-I inActivation of the Phosphatidylinositol 3-Kinase Pathway and CardiacChamber Morphogenesis,” J Biol Chem 274(52):37362-37369 (1999);Rentschler et al., “Visualization and Functional Characterization of theDeveloping Murine Cardiac Conduction System,” Development128(10):1785-1792 (2001); Rentschler et al., “Neuregulin-1 PromotesFormation of the Murine Cardiac Conduction System,” Proc Natl Acad SciUSA 99(16):10464-10469 (2002); Ruhparwar et al., “Prospects forBiological Cardiac Pacemaker Systems,” Pacing Clin Electrophysiol26(11):2069-2071 (2003)), endothelin (Gourdie et al.,“Endothelin-Induced Conversion of Embryonic Heart Muscle Cells IntoImpulse-Conducting Purkinje Fibers,” Proc Natl Acad Sci USA95(12):6815-6818 (1998); Hyer et al., “Induction of Purkinje FiberDifferentiation By Coronary Arterialization,” Proc Natl Acad Sci USA96(23):13214-13218 (1999); Cheng et al., “Development of the CardiacConduction System Involves Recruitment Within a MultipotentCardiomyogenic Lineage,” Development 126(22):5041-5049 (1999)), retinoicacid (Xavier-Neto et al., “A Retinoic Acid-Inducible Transgenic Markerof Sino-Atrial Development in the Mouse Heart,” Development126(12):2677-2687 (1999); Rosenthal et al., “From the Bottom of theHeart: Anteroposterior Decisions in Cardiac Muscle Differentiation,”Curr Opin Cell Biol 12(6):742-746 (2000); Xavier-Neto et al., “RetinoidSignaling and Cardiac Anteroposterior Segmentation,” Genesis31(3):97-104 (2001); Hochgreb et al., “A Caudorostral Wave of RALDH2Conveys Anteroposterior Information to the Cardiac Field,” Development130(22):5363-5374 (2003)) and Wnt family ligands (Bond et al., “Wnt11and Wnt7a Are Up-Regulated in Association With Differentiation ofCardiac Conduction Cells In Vitro and In Vivo,” Dev Dyn 227(4):536-543(2003); Tabibiazar et al., “Transcriptional Profiling of the HeartReveals Chamber-Specific Gene Expression Patterns,” Circ Res93(12):1193-1201 (2003); Monaghan et al., “Dickkopf Genes areCo-ordinately Expressed in Mesodermal Lineages,” Mech Dev 87(1-2):45-56(1999)). Several recent reviews have presented detailed descriptions ofthe development of the cardiac pacemaker and conduction systems (Gourdieet al., “Development of the Cardiac Pacemaking and Conduction System,”Birth Defects Res Part C Embryo Today 69(1):46-57 (2003); Christoffelset al., “Architectural Plan for the Heart: Early Patterning andDelineation of the Chambers and the Nodes,” Trends Cardiovasc Med14(8):301-307 (2004)). Broadly speaking, there are three fundamentalevents in their formation: First, a population of cardiomyocytes in theposterior, sinoatrial pole of the early embryonic heart tube assumes anodal/pacemaker-like phenotype and acts as a dominant pacemaker, drivingrhythmic propagation along the posterior-to-anterior axis (Kamino K,“Optical Approaches to Ontogeny of Electrical Activity and RelatedFunctional Organization During Early Heart Development,” Physiol Rev71(1):53-91 (1991); Satin et al., “Development of Cardiac Beat Rate inEarly Chick Embryos is Regulated By Regional Cues,” Dev Biol129(1):103-113 (1988)). Retinoid signaling has been implicated asimportant in establishing this polarity (Xavier-Neto et al., “A RetinoicAcid-Inducible Transgenic Marker of Sino-Atrial Development in the MouseHeart,” Development 126(12):2677-2687 (1999); Rosenthal et al., “Fromthe Bottom of the Heart: Anteroposterior Decisions in Cardiac MuscleDifferentiation,” Curr Opin Cell Biol 12(6):742-746 (2000); Xavier-Netoet al., “Retinoid Signaling and Cardiac Anteroposterior Segmentation,”Genesis 31(3):97-104 (2001); Hochgreb et al., “A Caudorostral Wave ofRALDH2 Conveys Anteroposterior Information to the Cardiac Field,”Development 130(22):5363-5374 (2003)). Second, while conduction velocitygenerally increases along the looping heart tube, specialized regions(specifically, the AV canal, sinoatrial pole, and outflow tract) retainthe comparatively slow propagation of the early tube heart (de Jong etal., “Persisting Zones of Slow Impulse Conduction in Developing ChickenHearts,” Circ Res 71(2):240-250 (1992); Arguello et al.,“Electrophysiological and Ultrastructural Study of the AtrioventricularCanal During the Development of the Chick Embryo,” J Mol Cell Cardiol18(5):499-510 (1986)). Despite its importance, surprisingly little isknown about the identity of the inductive cues driving this particulardevelopment. It results in sequential activation of the atrial andventricular segments and may improve the pumping efficiency of the hearttube, because the slowly-conducting segments function as sphincter-likevalves. Third, around the time of chamber septation, the fast cardiacconduction system (i.e., the His-Purkinje system) is recruited from“working” ventricular myocytes. Two factors released by endothelialcells have been implicated in this induction, endothelin (Gourdie etal., “Endothelin-Induced Conversion of Embryonic Heart Muscle Cells IntoImpulse-Conducting Purkinje Fibers,” Proc Natl Acad Sci USA95(12):6815-6818 (1998); Hyer et al., “Induction of Purkinje FiberDifferentiation By Coronary Arterialization,” Proc Natl Acad Sci USA96(23):13214-13218 (1999); Cheng et al., “Development of the CardiacConduction System Involves Recruitment Within a MultipotentCardiomyogenic Lineage,” Development 126(22):5041-5049 (1999)) andneuregulin (NRG) (Rentschler et al., “Visualization and FunctionalCharacterization of the Developing Murine Cardiac Conduction System,”Development 128(10):1785-1792 (2001)).

While the precise details regarding the timing and identity of inductivecues vary by species, this general sequence of events (early fatedecision regarding pacemaker vs. working atrial or ventricularcardiomyocyte→emergence of specialized slower-conducting nodalcenters→Purkinje-His conduction system differentiation) appearsconserved across vertebrate cardiac morphogenesis.

Neuregulin/ErbB Signaling in Cardiac Development and SubtypeSpecialization.

The neuregulins (NRG1-4) are members of the epidermal growth factorfamily that exert diverse biologic effects via the tyrosine receptorkinases ErbB2, ErbB3, and ErbB4. Very little is known about thefunctions of NRG2-4, but signaling by the NRG1 ligand is known to serveimportant and diverse functions in both cardiac development andpostnatal function (for a comprehensive review of its role in the adultheart, see Negro et al., “Essential Roles of Her2/erbB2 in CardiacDevelopment and Function,” Recent Prog Horm Res 59:1-12 (2004); Lemmenset al., “Role of Neuregulin-1/ErbB Signaling in CardiovascularPhysiology and Disease: Implications for Therapy of Heart Failure,”Circulation 116(8):954-960 (2007); Garratt et al., “ErbB2 Pathways inHeart and Neural Diseases,” Trends Cardiovasc Med 13(2):80-86 (2003);Iwamoto et al., “ErbB and HB-EGF Signaling in Heart Development andFunction,” Cell Struct Funct 31(1):1-14 (2006)). NRG1/ErbB signaling isremarkably complex: the 1.4 Mb NRG1 gene encodes for at least 15 ligandisoforms, which are classified based on their type of EGF domain (“α” or“β”) and N-terminal region (types I, II, or III). Importantly,recombinant peptides consisting of the EGF-like domain (i.e., NRG1-α or-β) alone are sufficient to activate the appropriate ErbB receptors andhave been used in most studies to date. All bioactive NRG1 ligands canbind to both ErbB3 and ErbB4 receptors, which results in receptor homo-or heterodimerization and activation of diverse downstream signaltransduction cascades (Falls D L, “Neuregulins: Functions, Forms, andSignaling Strategies,” Exp Cell Res 284(1):14-30 (2003); Holbro et al.,“ErbB Receptors: Directing Key Signaling Networks Throughout Life,” AnnuRev Pharmacol Toxicol 44:195-217 (2004)). Adding further complexity,there is another ErbB family member, the “orphan” receptor ErbB2, thatlacks ligand binding capacity, but can mediate downstream signaling uponactivation via heterodimerization with ErbB3 or ErbB4. Reciprocally,ErbB3 can bind NRG1 but lacks intrinsic kinase activity, and so thisisoform must also heterodimerize to function.

Work in the murine model has demonstrated the importance of NRG1/ErbBsignaling in early cardiac development. In the murine embryonic tubeheart (likely a state of maturation similar to hESC-CMs), NRG1 isstrongly expressed by the ventricular endocardium (and weakly by theatrial endocardium) (Meyer et al., “Multiple Essential Functions ofNeuregulin in Development,” Nature 378(6555):386-390 (1995); Kramer etal., “Neuregulins With an Ig-Like Domain Are Essential For MouseMyocardial and Neuronal Development,” Proc Natl Acad Sci USA93(10):4833-4838 (1996); Corfas et al., “Differential Expression of ARIAIsoforms in the Rat Brain,” Neuron 14(1):103-115 (1995); Zhao et al.,“Selective Disruption of Neuregulin-1 Function in Vertebrate EmbryosUsing Ribozyme-tRNA Transgenes,” Development 125(10):1899-1907 (1998)),while adjacent cardiomyocytes express ErbB2 and ErbB4 receptors but notNRG1 ligand (Meyer et al., “Multiple Essential Functions of Neuregulinin Development,” Nature 378(6555):386-390 (1995); Gassmann et al.,“Aberrant Neural and Cardiac Development in Mice Lacking the ErbB4Neuregulin Receptor,” Nature 378(6555):390-394 (1995); Lee et al.,“Requirement for Neuregulin Receptor ErbB2 in Neural and CardiacDevelopment,” Nature 378(6555):394-398 (1995)). Of note, NRG1-, ErbB2-,and ErbB4-null mice all die midway through embryogenesis (˜embryonic day10) with an almost identical cardiac malformation, a failure to formventricular trabeculae (Meyer et al., “Multiple Essential Functions ofNeuregulin in Development,” Nature 378(6555):386-390 (1995); Kramer etal., “Neuregulins With an Ig-Like Domain Are Essential For MouseMyocardial and Neuronal Development,” Proc Natl Acad Sci USA93(10):4833-4838 (1996); Corfas et al., “Differential Expression of ARIAIsoforms in the Rat Brain,” Neuron 14(1):103-115 (1995); Zhao et al.,“Selective Disruption of Neuregulin-1 Function in Vertebrate EmbryosUsing Ribozyme-tRNA Transgenes,” Development 125(10):1899-1907 (1998);Gassmann et al., “Aberrant Neural and Cardiac Development in MiceLacking the ErbB4 Neuregulin Receptor,” Nature 378(6555):390-394(1995)). These results indicate a requirement for NRG1/ErbB signaling,likely via ErbB2/ErbB4 heterodimers, in this very early event inventricular maturation. Interestingly, this defect in cardiacdevelopment is phenocopied in mice deficient in the fast sodium channel(SCN5A) (Papadatos et al., “Slowed Conduction and VentricularTachycardia After Targeted Disruption of the Cardiac Sodium Channel GeneScn5a,” Proc Natl Acad Sci USA 99(9):6210-6215 (2002)), a finding whichhas led to speculation that electrophysiological abnormalities in theNRG1/ErbB mutants may be responsible for the latter's phenotype (GarrattA N, ““To Erb-B or Not to Erb-B . . . .” Neuregulin-1/ErbB Signaling inHeart Development and Function,” J Mol Cell Cardiol 41(2):215-218(2006)).

NRG1/ErbB signaling has been implicated in the differentiation of thespecialized cardiac conduction system. Prior work in the avian model hadimplicated another endothelial-released cytokine, endothelin, asinducing Purkinje fiber specialization by “working” ventricularcardiomyocytes. To determine whether endothelin or other factors mightmediate similar effects in the developing mouse heart, Rentschler etal., “Visualization and Functional Characterization of the DevelopingMurine Cardiac Conduction System,” Development 128(10):1785-1792 (2001),employed the ‘CCS-lacZ’ (i.e., engrailed lacZ) transgenic mouse in whichβ-galactosidase is expressed in the cardiac conduction system. Usingthis convenient readout in an organ culture model, these authors foundthat, while application of endothelin had no effect, NRG1 greatlyexpanded β-galactosidase-expressing cardiac conduction system structuresin an mouse organ culture model (Rentschler et al., “Neuregulin-1Promotes Formation of the Murine Cardiac Conduction System,” Proc NatlAcad Sci USA 99(16):10464-10469 (2002)). Subsequently, a number ofgroups have looked for similar effects in primary or murine ESC-derivedembryonic cardiomyocytes with conflicting results, variably reportingthat pacemaker or conduction system differentiation can be induced byNRG1 (Ruhparwar et al., “Enrichment of Cardiac Pacemaker-Like Cells:Neuregulin-1 and Cyclic AMP Increase I(f)-Current Density and Connexin40 mRNA Levels in Fetal Cardiomyocytes,” Med Biol Eng Comput45(2):221-227 (2007)), both endothelin and NRG1 (Patel et al.,“Endothelin-1 and Neuregulin-1 Convert Embryonic Cardiomyocytes IntoCells of the Conduction System in the Mouse,” Dev Dyn 233(1):20-28(2005)), or endothelin but not NRG1 (Gassanov et al., “EndothelinInduces Differentiation of ANP-EGFP Expressing Embryonic Stem CellsTowards a Pacemaker Phenotype,” Faseb J 18(14):1710-1712 (2004)). Onecaveat related to the latter studies is that they have sometimes tendedto blur endpoints for cardiac pacemaker (e.g., sinoatrial) andconduction (e.g., Purkinje) system differentiation, which, as discussedin the preceding section, are known to be spatially and temporallydistinct events in cardiac development. This may account in part fortheir ostensibly different conclusions.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method ofproducing cardiomyocytes having a nodal/pacemaker phenotype. This methodinvolves culturing stem cells under conditions effective to producecardiomyocytes and contacting the cardiomyocytes with an antagonist ofneuregulin-1 or an antagonist of ErbB under conditions effective toinduce the production of cardiomyocytes having a nodal/pacemakerphenotype

A second aspect of the present invention is directed to an isolatedpopulation of nodal/pacemaker cardiomyocytes. This isolated populationof cardiomyocytes is characterized by a spontaneous firing rate of >90bpm and a slow, biphasic action potential upstroke of <15V/s.

A third aspect of the present invention is directed to a method oftreating cardiac arrhythmia in a subject. This method involves providingan isolated population of nodal/pacemaker cardiomyocytes of the presentinvention and delivering the isolated population of nodal/pacemakercardiomyocytes to the subject under conditions effective to treat thecardiac arrhythmia.

A fourth aspect of the present invention is directed to a method ofproducing cardiomyocytes having an atrial/ventricular phenotype. Thismethod involves culturing stem cells under conditions effective toproduce cardiomyocytes and contacting the cardiomyocytes with aneuregulin-1 agonist, neuregulin-1 mimetic, or a related agonist of anErbB receptor under conditions effective to induce the production ofcardiomyocytes having an atrial/ventricular phenotype.

A fifth aspect of the present invention is directed to an isolatedpopulation of atrial/ventricular cardiomyocytes.

A sixth aspect of the present invention is directed to a method ofimproving cardiac tissue repair or cardiac organ function in a subject.This method involves providing an isolated population ofatrial/ventricular cardiomyocytes of the present invention anddelivering the isolated atrial/ventricular cardiomyocytes to the subjectunder conditions effective to improve cardiac tissue repair or cardiacorgan function.

Cell-based therapies have tremendous promise in ameliorating or evenreversing serious cardiac diseases. Still, it will almost certainlyprove insufficient to merely differentiate stem cells into admixedcardiomyocytes. To ensure proper host-graft integration and attenuatethe risk of arrhythmogenesis following cell transplantation, the fieldwill need to guide the differentiation of stem cells in the correctcardiac subtypes for any given clinical application. The presentinvention is responsive to this need by providing a means to deriveenriched populations of nodal/pacemaker cells (e.g., for application asa biological pacemaker) or working, chamber-specific cardiomyocytes(e.g., for replacement of lost ventricular muscle following an infarct).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show hESC-derived cardiomyocytes exhibit distinct nodal andworking-type action potentials. Spontaneous action potentials (“APs”)exhibited by two representative hESC-CMs showing characteristicnodal—(FIG. 1A) and working-type (FIG. 1B) AP waveforms and parameters.AP recordings were obtained from isolated cardiomyocytes at 36±1° C.,using the perforated-patch clamp technique.

FIGS. 2A-2D show that activation of the cGATA6-EGFP transgene identifieshESC-derived cardiomyocytes with a nodal phenotype. hESC-CM cultureswere transduced with a lentiviral vector in which the nodal-specificcGATA6 promoter drives expression of EGFP. About 50% of the cells weretransduced, and, by 72 hours post-transduction, ˜15% of the total cellpopulation showed EGFP expression. cGATA6-EGFP+ cells expressed thecardiac marker troponin T (cTnT) (FIG. 2A, left panel) and the pacemakerion channel HCN4 (FIG. 2B, left panel). EGFP expression was confirmedusing an anti-EGFP antibody (FIGS. 2A and 2B, middle panels). An overlayof cTnT or HCN4 expression with EGFP is shown in the right panels ofFIGS. 2A and 2B, respectively. Scalebar=20 μm. FIG. 2C is aphotomicrograph showing a patch-clamp electrode (arrow, upper panel) ona representative EGFP+ cell, and its corresponding nodal-type APrecording (lower panel). 20 of 21 EGFP+ hESC-CMs showed a nodal-like APphenotype, versus only 2 of 20 EGFP− myocytes. FIG. 2D shows histogramplots indicating the number of EGFP+ (top plot) and EGFP− (bottom plot)hESC-CMs exhibiting any given maximal upstroke velocity (dV/dtmax).While virtually all of the EGFP+ putative nodal cells had a dV/dtmax<15V/s, the EGFP− cells (which likely include both working-type andnon-transduced cells) exhibited a wide range of dV/dtmax values.

FIGS. 3A-3D demonstrate that hESC-derived cardiomyocytes exhibit anintact NRG-1/ErbB signaling pathway. RT-PCR analysis confirmingexpression of the α and β isoforms of NRG-1, as well as ErbB2, ErbB3,and ErbB4 receptors in both undifferentiated hESC and hESC-CM culturesis shown in FIG. 3A. Adult human heart and human umbilical veinendothelial cells (HUVECs) were examined as positive controls. FIGS. 3Band 3C are immunofluorescence images of representative hESC-CMs, all ofwhich expressed β-myosin heavy chain (β-MHC) (FIGS. 3B and 3C, leftpanels) as well as ErbB2 (FIG. 3B, middle panel) and ErbB4 (FIG. 3C,middle panel). Relatively few non-cardiac cells (arrow) showed ErbBexpression. Bar=25 μm. Immunoblots for phosphorylated and total Akt andERK (p42 and p44 isoforms) kinases in hESC-CM cultures are shown in FIG.3D. Treatment with NRG-1β agonist (“NRG ligand”) resulted in theactivation of both kinases, an effect that was inhibited by simultaneoustreatment with an anti-NRG-1β-neutralizing antibody (“anti-NRG”). Allimages are representative of at least 3 biological replicates.

FIGS. 4A-4C demonstrate that interference with NRG-1/ErbB signalingchanges the ratio of nodal versus working type cells in differentiatinghESC-derived cardiomyocyte cultures. FIG. 4A illustrates the protocolsused to generate hESC-CMs under control or NRG-1/ErbB manipulatedconditions. AA=activin A. FIG. 4B shows the percentage of cardiomyocytesexhibiting the nodal AP phenotype in hESC-CM cultures treated withcontrol, non-immune IgG (25 mg/ml, n=33 cells), DMSO vehicle (0.1%, n=24cells), anti-NRG-1β neutralizing antibody (25 mg/ml, n=38 cells), ErbBreceptor antagonist AG1478 (10 mM, n=21 cells), or exogenous NRG-1βagonist (100 ng/ml) during day 5 to 12 (n=28 cells) or continuouslyafter day 5 (n=21 cells). Note that interference with NRG-1/ErbBsignaling greatly enhanced the proportion of nodal cells relative tocontrol or NRG-1β treated conditions. In FIG. 4C, the precedingexperiment was repeated using cGATA6-EGFP− transduced cultures, andplotted are the percentage of EGFP+ putative nodal cells generated undercontrol or NRG-1/ErbB manipulated conditions (n=4 biologicalreplicates). Note that, when corrected for the ˜50% transductionefficiency, the proportion of nodal cells under each conditionapproximates that previously determined by direct AP phenotyping. Groupswere compared by Fisher's exact test with Bonferroni correction.*P<0.05, **P<0.01.

FIG. 5 shows that interference with NRG-1/ErbB signaling changes theexpression of cardiac subtype-specific genes. Quantitative RT-PCRanalysis of cardiac subtype-specific genes showing differentialexpression in control, AG1478-, or exogenous NRG-1b-treated hESC-CMcultures. Transcript levels are shown normalized to that in controls.Groups were compared by Bonferroni corrected one-way ANOVA with †P<0.05,‡P<0.01 vs. control, *P<0.05, **P<0.01 vs. NRG-treated. Labels indicatethe anticipated pattern of expression in nodal- and working-type cells.See Table 4 for results from the full panel of genes examined, includingthose which did not show statistically significant changes.

FIGS. 6A-6B demonstrate that cGATA6-EGFP positive cells always expresscardiac markers. The left panel of FIG. 6A shows representativeimmunofluorescence images of EGFP+ cells expressing the striatedmuscle-marker sarcomeric actin (sActin). EGFP expression is shown in themiddle panel of FIG. 6A and a merged image showing co-localization ofsActin and EGFP expression in depicted in the right panel of FIG. 6A. InFIG. 6B, a differential interference contrast (DIC) picture combinedwith the fluorescence of EGFP (left panel) and the image stained withβ-myosin heavy chain (β-MHC, right panel) from the same cells shows thatall EGFP+ cells are cardiomyocytes. Scalebar=20 μm.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is directed to a method ofproducing cardiomyocytes having a nodal/pacemaker phenotype. This methodinvolves culturing stem cells under conditions effective to producecardiomyocytes and contacting the cardiomyocytes with an antagonist ofneuregulin-1 or an antagonist of ErbB under conditions effective toinduce the production of cardiomyocytes having a nodal/pacemakerphenotype

In accordance with this aspect of the present invention, “stem cells”encompass cells which have the ability to proliferate and form cells ofmore than one different phenotype, and are further capable of selfrenewal, either as part of the same culture or when cultured underdifferent conditions. Stem cells suitable for use in the methods of thepresent invention include embryonic stem cells or germ cells, adult stemcells, and induced pluripotent stem cells.

Embryonic stem (“ES”) cells include any multi- or pluripotent stem cellderived from pre-embryonic, embryonic, or fetal tissue at any time afterfertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art acceptedtest (e.g., the ability to form a teratoma in 8-12 week old SCID mice).In a preferred embodiment of the present invention, the stem cells aremammalian embryonic stem cells. More preferably, the embryonic stemcells of the present invention are human embryonic stem cells.

Methods for culturing embryonic stems cells, particularly humanembryonic stem cells, are known in the art and described inWO2006/029297, WO2006/019366 and WO2006/029198 all to Thomson andLudwig, and WO2008/089351 to Bergendahl and Thomson, which are herebyincorporated by reference in their entirety.

Embryonic germ (“EG”) cells are derived from primordial germ cells andexhibit an embryonic pluripotent cell phenotype. EG cells are capable ofdifferentiation into cells of ectodermal, endodermal, and mesodermalgerm layers. EG cells can also be characterized by the presence orabsence of markers associated with specific epitope sites. Methods forisolating, culturing, and characterizing human EG cells are described inShamblott et al., “Human Embryonic Germ Cell Derivatives Express a BroadRange of Developmentally Distinct Markers and Proliferate Extensively InVitro,” Proc Natl Acad Sci 98(1):113-118 (2001), which is herebyincorporated by reference in its entirety.

Adult stem cells, as used in accordance with the present invention,encompass cells that are derived from any adult tissue or organ thatreplicate as undifferentiated cells and have the potential todifferentiate into at least one, preferably multiple, cell lineages.General methods for producing and culturing populations of adult stemcells suitable for use in the present invention are described inWO2006/110806 to Xu et al., WO2002/057430 to Escoms et al., andWO2006/112365 to Nagaya, which are hereby incorporated by reference intheir entirety. Cardiac progenitor or adult stem cells are particularlysuitable for use in the present invention. Methods for isolating andculturing cardiac stem cells are described in WO2007/100530,WO2002/009650, and WO2002/013760 all to Anversa; WO2004/019767 toSchneider; and WO2006/052925 to Marban et al., which are all herebyincorporated by reference in their entirety.

Induced pluripotent stem cells (“iPSC”) are also suitable for use in themethods of the present invention. iPSCs, as used herein, refer topluripotent stem cells induced from somatic cells, e.g., a population ofdifferentiated somatic cells (Takahashi et al., “Induction ofPluripotent Stem Cells From Adult Human Fibroblasts By Defined Factors,”Cell 131(5):861-872 (2007); Park et al., “Reprogramming of Human SomaticCells to Pluripotency With Defined Factors,” Nature (2007); and Yu etal., “Induced Pluripotent Stem Cell Lines Derived From Human SomaticCells,” Science 318(5858):1917-1920 (2007), which are herebyincorporated by reference in their entirety). iPSCs are capable ofself-renewal and differentiation into cell fate-committed stem cells,including various types of mature cells. iPSCs exhibit normalmorphological (i.e., round shape, large nucleoli and scant cytoplasm)and growth properties, and express pluripotent cell-specific markers(e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, but not SSEA-I). iPSCsare substantially genetically identical to their respectivedifferentiated somatic cells of origin, yet display characteristicssimilar to higher potency cells, such as ES cells. iPSCs can be obtainedfrom various differentiated (i.e., non-pluripotent and multipotent)somatic cells. Although various somatic cells are suitable for iPSCinduction, higher reprogramming frequencies are observed when thestarting somatic cells have a doubling time of about twenty-four hours.Somatic cells useful for carrying out the methods of the presentinvention include non-embryonic cells obtained from a fetal, newborn,juvenile or adult primates. Preferably, the somatic cells are humansomatic cells. Examples of somatic cells include, but are not limitedto, bone marrow cells, epithelial cells, fibroblast cells, hematopoieticcells, hepatic cells, intestinal cells, mesenchymal cells, myeloidprecursor cells and spleen cells. Other somatic cells suitable for usein the present invention include CD29⁺ CD44⁺ CD166⁺ CD105⁺ CD73⁺ andCD31⁺ mesenchymal cells that attach to a substrate. Alternatively, thesomatic cells can be cells that themselves proliferate and differentiateinto other types of cells, including blood stem cells, muscle/bone stemcells, brain stem cells, and liver stem cells. Multipotent hematopoieticcells, including myeloid precursor or mesenchymal cells, are alsosuitable for use in the methods of the invention. Methods for producingand culturing populations of iPSCs are described in WO2008/118820 toThomson and Yu and WO2007/069666 to Yamanaka, which are herebyincorporated by reference in their entirety.

In accordance with this aspect of the present invention, the stem cellsare cultured under conditions effective to induce cardiomyocytedifferentiation. As described herein, the preferred method for directingcardiomyocyte differentiation relies on sequential treatment ofundifferentiated stem cells with activin-A and bone morphogenicprotein-4 (BMP4) as described in WO2007/002136 and WO2005/090558 both toGold et al.; WO2003/006950 to Xu; and Laflamme et al., “CardiomyocytesDerived from Human Embryonic Stem Cells in Pro-Survival Factors EnhanceFunction of Infarcted Rat Hearts,” Nat Biotechnol 25(9):1015-1024(2007), which are hereby incorporated by reference in their entirety.This method reliably produces large quantities of relatively purepopulations (˜90%) of cardiomyocytes. The differentiated cardiomyocytescan be further contacted with a cocktail of pro-survival factors (e.g.,Matrigel, Bcl-XL, cyclosporine A, a compound that opens ATP-dependent K+channels, IGF-1, and the caspase inhibitor ZVAD-fmk) to limit cell deathand promote survival upon transplantation to the target heart tissue.

Various other methods for directing cardiomyocyte differentiation thatare known in the art are also suitable for use in the present invention(see e.g., WO2007/038933 to Rigshospitalet; WO2008/088882 to Bruneau andTakeuchi; WO2003/035838 to Epstein et al.; WO2004/081205 to Mummery etal.; WO2005/118784 to Mummery and Passier; WO2006/066320 to Passier andMummery; and WO2007/070964 to Davidson et al., which are herebyincorporated by reference in their entirety).

Once a population of cardiomyocytes are obtained, inducing anodal/pacemaker phenotype in these cells involves administering to thecardiomyocytes an antagonist of neuregulin-1 or an antagonist of ErbB.As used herein, “antagonist” broadly encompasses any agent that inhibitsneuregulin-1 expression, activity, and/or signaling and any agent thatinhibits ErbB receptor expression, activity, and/or signaling. Theneuregulin-1 or ErbB antagonist of the present invention preferablyinhibits neuregulin-1/ErbB mediated activity and signaling in thecardiomyocytes. In a preferred embodiment of the present invention, theantagonist of neuregulin-1 is an antagonist (i.e., inhibitor) ofneuregulin-1β. Suitable antagonists of both neuregulin-1 and ErbBinclude nucleic acid molecule inhibitors, protein or peptide inhibitors,or small molecule inhibitors.

Suitable nucleic acid molecule inhibitors of neuregulin-1 and ErbBinclude antisense RNAs or RNAi, such as short interfering RNAs (siRNA),short hairpin RNAs (shRNA), microRNAs, and aptamers.

As described herein, methods of designing and making antisense RNAmolecules, RNAi molecules, microRNAs and aptamers are well known in theart, and facilitated by knowledge of the nucleic acid sequence of thetarget molecule to be inhibited, in this case neuregulin-1 and/or ErbB.The nucleic acid sequence of human neuregulin-1 and its related isoformshave been described and are readily available to one of skill in the art(see Genbank Accession Nos. NM_(—)004495.2 (neuregulin-1 isoform HRGγ);NM_(—)013956.2 (neuregulin-1 isoform HRG-β1); NM_(—)013957.2(neuregulin-1 isoform HRG β2); NM_(—)013958.2 (neuregulin-1 isoform HRGβ3); NM_(—)013959.2 (neuregulin-1 isoform SMDF); NM_(—)013960.2(neuregulin-1 isoform ndf43); NM_(—)013961.2 (neuregulin-1 isoform GGF);NM_(—)013962.2 (neuregulin-1 isoform GGF2); NM_(—)013964.2 (neuregulin-1isoform HRG-α), which are all hereby incorporated by reference in theirentirety). Likewise, the nucleic acid sequences of the various humanErbB receptors are also known in the art (see Genbank Accession Nos.NM_(—)004448 (ErbB2); NM_(—)001982 (ErbB3); and NM_(—)005235 (ErbB4),which are all hereby incorporated by reference in their entirety).

Methods of making antisense molecules and their use to inhibit the invitro and in vivo translation of genes is well known in the art (seee.g., U.S. Pat. Nos. 7,425,544 to Dobie et al.; 7,307,069 to Karras etal.; 7,288,530 to Bennett et al.; 7,179,796 to Cowsert et al.; 6,277,640to Benett et al.; 6,255,111 to Bennett et al; and U.S. PatentPublication No. 20050130927 to Karl-Hermann et al., which are all herebyincorporated by reference in their entirety). Antisense nucleic acidsare nucleic acid molecules (e.g., molecules containing DNA nucleotides,RNA nucleotides, or modifications (e.g., modification that increase thestability of the molecule, such as 2′-O-alkyl (e.g., methyl) substitutednucleotides) or combinations thereof) that are complementary to, or thathybridize to, at least a portion of a specific nucleic acid molecule,such as an RNA molecule (e.g., an mRNA molecule) (see e.g., Weintraub HM, “Antisense RNA and DNA,” Scientific American 262:40 (1990), which ishereby incorporated by reference in its entirety). The antisense nucleicacid molecules hybridize to corresponding nucleic acids, such as mRNAs,to form a double-stranded molecule, which interferes with translation ofthe mRNA, as the cell will not translate a double-stranded mRNA.Antisense nucleic acids used in the invention are typically at least10-12 nucleotides in length, for example, at least 15, 20, 25, 50, 75,or 100 nucleotides in length. The antisense nucleic acid molecules canalso be as long as the target nucleic acid with which it is intended toform an inhibitory duplex. In a preferred embodiment of the presentinvention, antisense molecules targeting neuregulin-1 inhibit or reducethe expression of neuregulin-1β. In another embodiment, antisensemolecules targeting ErbB, inhibit or reduce the expression of ErbB2,ErbB3, ErbB4, or a combination thereof. Antisense molecules directed tohuman ErbB2 are described in U.S. Pat. Nos. 6,365,345 to Brysch et al.;5,968,748 to Bennett et al.; and U.S. Patent Publication No. 20050130927to Karl-Hermann et al., which are hereby incorporated by reference intheir entirety. Antisense molecules directed to human ErbB3 and ErbB4are described in U.S. Pat. Nos. 6,277,640 and 6,255,111, respectively,both to Bennett et al., which are hereby incorporated by reference intheir entirety. Antisense nucleic acids can be introduced into cells asantisense oligonucleotides, or can be produced in a cell in which anucleic acid encoding the antisense nucleic acid has been introduced,for example, using gene therapy methods.

siRNAs are double stranded synthetic RNA molecules approximately 20-22nucleotides in length with short 2-3 nucleotide 3′ overhangs on bothends. The double stranded siRNA molecule represents the sense andanti-sense strand of a portion of the target mRNA molecule, in this casea portion of the neuregulin-1 or ErbB mRNA sequence. siRNA molecules aretypically designed to target a region of the mRNA target approximately50-100 nucleotides downstream from the start codon. Upon introductioninto a cell, the siRNA complex triggers the endogenous RNA interference(RNAi) pathway, resulting in the cleavage and degradation of the targetmRNA molecule. A number of exemplary siRNA molecules designed to targetthe human neuregulin-1 gene and isoforms thereof that are suitable foruse in accordance with this aspect of the present invention are known inthe art and are commercially available (e.g., Applied Biosystems, FosterCity, Calif. and Abnova, Walnut, Calif.). Likewise, meroduplex RNA(mdRNA) molecules that interfere with neuregulin-1 gene expression viathe cellular RNA interference machinery, are also useful in the methodsof the present invention (WO2008/109555 to Quay et al., which is herebyincorporated by reference in its entirety).

siRNA molecules directed to the ErbB family of tyrosine kinase receptorsare also well known in the art. In accordance with this aspect of theinvention, an siRNA molecule that interferes with the expression ofErbB2, ErbB3, ErbB4, or any combination thereof is suitable for use. Anumber of exemplary siRNA molecules targeting Erb2 expression aredescribed by Sahin et al., “Combinatorial RNAi for Quantitative ProteinNetwork Analysis,” Proc Natl Acad Sci USA. 104(16):6579-6584 (2007),which is hereby incorporated by reference in its entirety, including5′-PCAUUGUGCAGAAUUCGUCCUU (SEQ ID NO:1); 5′-PCCAUUGUGCAGAAUUCGUCUU (SEQID NO:2); 5′-PAAACGUGUCUGUGUUGUAGUU (SEQ ID NO:3); and5′-PCAUCACGUAUGCUUCGUCUUU (SEQ ID NO:4). An exemplary diced-doublestranded siRNA molecule directed to nucleotides 1038-1531 of the humanErbB3 gene sequence is described by Tapinos et al., “ErbB2 ReceptorTyrosine Kinase Signaling Mediates Early Demyelination Induced byLeprosy Bacilli,” Nat Med. 12(8):961-966 (2006), which is herebyincorporated by reference in its entirety. siRNA molecules targetingErbB4 expression that can be used in accordance with this aspect of thepresent invention include 5′-ACUGAGCUCUCUCUCUGACTT-3′(SEQ ID NO:6) and5′-GUCAGAGAGAGAGCUCAGUTT-3′(SEQ ID NO:7) (Maatta et al., “ProteolyticCleavage and Phosphorylation of a Tumor-Associated ErbB4 Isoform PromoteLigand-Independent Survival and Cancer Cell Growth,” Mol Biol Cell.17(1):67-79 (2006), which is hereby incorporated by reference in itsentirety).

Various modifications to the above referenced siRNA compositions, suchas the incorporation of modified nucleosides or motifs into one or bothstrands of the siRNA molecule can be incorporated to enhance stability,specificity, and efficacy of the siRNA molecules. Such modifications arewell known in the art and have been described in WO2004/015107 to Gieseet al.; WO2003/070918 to McSwiggen et al.; WO1998/39352 to Imanishi etal.; U.S. Patent Application Publication No. 2002/0068708 to Jesper etal.; U.S. Patent Application Publication No. 2002/0147332 to Kaneko etal; U.S. Patent Application Publication No. 2008/0119427 to Bhat et al.,which are all hereby incorporated by reference in their entirety.

Short or small hairpin RNA (shRNA) molecules are similar to siRNAmolecules in function, but comprise longer RNA sequences that make atight hairpin turn. shRNA is cleaved by cellular machinery into siRNA,and like siRNA, they silence gene expression via the cellular RNAinterference pathway.

Nucleic acid aptamers are molecules that interact and bind to a targetmolecule (e.g., neuregulin-1β or ErbB) with a very high degree ofspecificity. Typically, aptamers are small nucleic acids ranging from15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules as well as large molecules, as described in U.S. Pat. Nos.5,631,146 to Szostak; 5,786,462 to Schneider; 5,543,293 to Schneider;and 5,580,737 to Polisky, which are all hereby incorporated by referencein their entirety. Aptamers can bind very tightly with K_(d)s for thetarget molecule of less than 10⁻¹² M. It is preferred that the aptamersbind the target molecule with a K_(d) less than 10⁻⁶. It is morepreferred that the aptamers bind the target molecule with a K_(d) lessthan 10⁻⁸.

Other nucleic acid molecules suitable for the inhibition of neuregulin-1or ErbB include ribozymes (U.S. Pat. No. 5,334,711 to Sproat et al; U.S.Pat. No. 5,646,031 to DeYoung et al.; U.S. Pat. No. 5,595,873 to Joyceet al.; U.S. Pat. No. 5,580,967 to Joyce et al., which are herebyincorporated by reference in their entirety), triplex forming functionalnucleic acid molecules (U.S. Pat. No. 5,176,996 to Hogan et al., whichis hereby incorporated by reference in its entirety) or external guidesequences (EGSs) (WO 92/03566 to Yale, which is hereby incorporated byreference in its entirety).

Protein or peptide antagonists of neuregulin-1 and ErbB expression oractivity are also suitable for use in accordance with this aspect of thepresent invention. Suitable protein inhibitors include, but are notlimited to, antibodies (e.g. single chain antibodies, chimericantibodies, hybrid antibodies), intrabodies, peptabodies, peptideaptamers, or any other binding molecules, including synthetic peptideinhibitors, having antigen binding specificity for neuregulin-1 or ErbB.The amino acid sequences encoding the human neuregulin-1 protein and itsisoforms and the various ErbB receptor tyrosine kinases are known in theart (see NCBI Protein Accession Nos. NP_(—)004486 (neuregulin-1 isoformHRG-γ); NP_(—)039250.2 (neuregulin-1 isoform HRGβ1); NP_(—)039251.2(neuregulin-1 isoform HRG-β2); NP_(—)039252.2 (neuregulin-1 isoformHRG-β3); NP_(—)039253.1 (neuregulin-1 isoform SMDF); NP_(—)039254.1(neuregulin-1 isoform ndf43); NP_(—)039255.1 (neuregulin-1 isoform GGF);NP_(—)039256.2 (neuregulin-1 isoform GGF2); NP_(—)039258.1 (neuregulin-1isoform HRG-α); NP_(—)00439.2 (human ErbB2); NP_(—)001973.2 (humanErbB3); NP_(—)005226.1 (human ErbB4), which are all hereby incorporatedby reference in their entirety), and, therefore, facilitate thegeneration of new protein or peptide antagonists using standardtechniques known in the art.

Preferred antibodies or other inhibitory binding molecules of thepresent invention are those having antigen specificity for neuregulin-1or ErbB, where upon binding of the antibody or binding molecule toneuregulin-1 or ErbB the expression and/or activity of neuregulin-1 orErbB is inhibited or significantly reduced. Antibodies of the presentinvention include monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), intrabodies, peptabodies, and antibodyfragments. Antibody fragments comprise a portion of a full lengthantibody, generally the antigen binding or variable domain thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

Antibodies having antigen specificity for neuregulin-1 are known in theart (see e.g. Trinidad et al., “The Agrin/MuSK Signaling Pathway isSpatially Segregated from the Neuregulin/ErbB Receptor Signaling Pathwayat the Neuromuscular Junction,” J Neuroscience 20(23):8762-70 (2000),which is hereby incorporated by reference in its entirety) and arecommercially available (e.g., Genetex, San Antonio, Tex.; R&D Systems,Minneapolis, Minn.; Abnova, Walnut, Calif.). In a preferred embodimentof the present invention, the neuregulin-1 antibody is a neutralizingantibody as described herein in the Examples.

Antibodies having antigen specificity to ErbB are also well known in theart (see e.g., Gilmour et al., “Neuregulin Expression, Function, andSignaling in Human Ovarian Cancer Cells,” Clin Cancer Res.8(12):3933-3942 (2002) (anti-ErbB3 antibody); Labriola et al.,“Heregulin Induces Transcriptional Activation of the ProgesteroneReceptor by a Mechanism that Requires Functional ErbB-2 andMitogen-Activated Protein Kinase Activation in Breast Cancer Cells,” MolCell Biol. 23(3):1095-1111 (2003) (anti-Erb4 antibody); Grim et al.,“ErbB-2 Knockout Employing an Intracellular Single-Chain Antibody (sFv)Accomplishes Specific Cytotoxicity in ErbB-2 Expressing Lung CancerCells,” Am J Resp Cell Mol Biol 15:348-354 (1996) (ErbB-2 single-chainantibody), U.S. Pat. No. 5,821,337 to Carter et al. (humanized ErbB-2antibody Herceptin); and U.S. Patent Publication No. 20060233808 toDeperthes (peptabodies to ErbB-1, -3, and -4), which are all herebyincorporated by reference in their entirety). Anti-ErbB antibodies arealso commercially available (e.g., Neomarkers/Lab Visions, Fremont,Calif.). Alternatively, polyclonal or monoclonal antibodies directed tohuman neuregulin-1 or ErbB can be obtained using standard techniques andprocedures known in the art for generating antibodies (see e.g.,ANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane eds., ColdSpring Harbor Laboratory, 1988) and LAWRENCE B. SHOOK, MONOCLONALANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, 51-63 (1987), which arehereby incorporated by reference in their entirety).

Other antibodies or protein or peptide binding molecules useful forcarrying out the methods of the present invention include those havingantigen binding specificity for a protein in the neuregulin-1 or ErbBsignal transduction pathway, where upon binding of the antibody or otherbinding molecule to its target protein, neuregulin-1 or ErbB function isindirectly disrupted.

Alternative protein inhibitors suitable for use in this aspect of thepresent invention include dominant negative forms of the neuregulin-1 orErbB proteins. U.S. Patent Application Publication No. 20050123538 toShemesh et al., which is hereby incorporated by reference in itsentirety, describes dominant negative forms of human ErbB2 which aredevoid of transmembrane and intracellular kinase domains, yet retaintheir ability to dimerize with other ErB receptors. These dominantnegative forms of ErbB2 sequester ErbB1, ErbB3, and ErbB4 proteins,thereby inhibiting their biological activity. Other peptide ligands,including both linear and cyclic peptide ligands that bind to andinhibit ErbB are described in U.S. Pat. No. 6,987,088 to Dennis, whichis hereby incorporated by reference in its entirety.

Other protein or peptide inhibitors of ErbB that are suitable for use inthe methods of the present invention include recombinant proteins orpeptide fragments thereof which mimic endogenous negative regulators ofErbB. For example, c-cbl and Nrdp 1 are ubiquitin ligases which controlErbB receptor degradation (Levkowitz et al., “Ubiquitin Ligase Activityand Tyrosine Phosphorylation Underlie Suppression or Growth FactorSignaling by c-Cble/Sli-1,” Mol Cell 4:1029-40 (1999); Diamonte et al.,“An RBCC Protein Implicated in Maintenance of Steady-State NeuregulinReceptor Levels,” Proc Natl Acad Sci USA 99:2866-2871 (2002); and Qiu etal., “Nrdp1/FLRF is a Ubiquitin Ligase Promoting Ubiquitination andDegradation of the Epidermal Growth Factor Receptor Family member,ErbB3,” Proc Natl Acad Sci USA 99:14843-48 (2002), which are herebyincorporated by reference in their entirety). Recombinant c-cbl or Nrdp1proteins or peptide fragments, or nucleic acid molecules encoding suchrecombinant proteins or peptides, can be utilized to enhance ErbBreceptor degradation thereby downregulating its activity. The nucleicacid and amino acid sequences of c-cbl (GenBank Accession No. CAA40393,which is hereby incorporated by reference in its entirety) and Nrdp1(NCBI Ref Seq Nos. NP_(—)005776 and NM_(—)005785, which are herebyincorporated by reference in it entirety) are well known in the art.Likewise, recombinant proteins or peptide fragments thereof, or nucleicacid molecules encoding the recombinant proteins or peptide, mimickingthe ErbB negative modulator proteins, herstatin, Argos, CPI, and Kek1(Azios et al., “Expression of Herstatin, an autoinhibitor of HER-2/neu,Inhibits Transactivation of Her-3 by Her-2 and Blocks EGF Activation ofthe EGF Receptor,” Oncogene 20:5199-5209 (2001); Blanco-Aparicio et al.,“Potato Carboxypeptidase Inhibitor, a T-Knot Protein, is an EpidermalGrowth Factor Antagonist that Inhibits Tumor Cell Growth,” J Biol Chem273:12370-12377 (1998); Ghiglione et al., “Mechanism of Inhibition ofthe Drosophila and Mammalian EGF Receptors by the Transmembrane ProteinKekkon-1,” Development 130:4483-4493 (2003); Sweeny et al., “NegativeRegulation of ErbB Family Receptor Tyrosine Kinases,” British J Cancer90:289-93 (2004), which are all hereby incorporated by reference intheir entirety), are also suitable for inhibiting ErbB mediated activityin accordance with the methods of the present invention. The nucleicacid and amino acid sequence of human herstatin is well known in the art(GenBank Accession No. AAD56009, which is incorporated by reference inits entirety).

Small molecule inhibitors suitable for use in accordance with thisaspect of the present invention include the pan-ErbB small moleculeinhibitors, JNJ-28871063 (Emanuel et al., “Cellular and In Vivo Activityof JNJ-28871063, a Nonquinazoline Pan-ErbB Kinase Inhibitor that Crossesthe Blood-Brain Barrier and Displays Efficacy Against IntracranialTumors,” Mol Pharmacol. 73(2):338-348 (2008), which is herebyincorporated by reference in its entirety) and CI-1033 (Slichenmyer etal., “CI-1033, a Pan-ErbB Tyrosine Kinase Inhibitor,” Semin Oncol. 28(5Suppl 16):80-85 (2001), which is hereby incorporated by reference in itsentirety). Likewise, the ErbB1/ErbB2 small molecule inhibitor PKI166(Brandt et al., “Mammary Glands Reconstituted with Neu/ErbB2 TransformedHC11 Cells Provide a Novel Orthotopic Tumor Model for TestingAnti-Cancer Agents,” Oncogene 20(39):5459-5465 (2001), which is herebyincorporated by reference in its entirety) is also suitable for use.Other small molecule inhibitors of ErbB that can be used in the methodsof the present invention include, tryphostin AG825, AG1478, PD158780,and BIBX1382B2 (see e.g., Egeblad et al., “BIBX1382BS, but Not AG1478 orPD153035, Inhibits the ErbB Kinases at Different Concentrations inIntact Cells,” Biochem Biophys Res Commun 281(1):25-21 (2001), which ishereby incorporated by reference in its entirety).

Another aspect of the present invention relates to an isolatedpopulation of nodal/pacemaker cardiomyocytes. In a preferred embodiment,the isolated population of nodal/pacemaker cardiomyocytes are humannodal/pacemaker cardiomyocytes derived from human stem cells inaccordance with the methods described herein. This isolated populationof human nodal/pacemaker cardiomyocytes is characterized by unambiguousmolecular and electrophysiological markers. Electrophysiological markersinclude a spontaneous firing rate of >90 bpm (mean of 122 bpm); a slow,biphasic action potential upstroke (dV/dtmax<15 V/s, mean of 6.5 V/s);robust pacemaker current under voltage clamp; and comparatively littleopposing IK1 current (see Table 1 below, all parameters acquired at 37°C.). Importantly, because these cells are true pacemaker/nodalcardiomyocytes, these cells retain the aforementionedelectrophysiological characteristics with maturation. The humanpacemaker/nodal cardiomyocytes also exhibit immunophenotypic markersincluding the usual pan-cardiac proteins (i.e., sarcomeric actin,sarcomeric myosin, troponins) as well the pacemaker channel HCN4 proteinexpression. Finally, the isolated population of nodal/pacemakercardiomyocytes of the present invention have reduced expression of“working” (i.e., atrial or ventricular chamber-specific) cardiac markersincluding KCNJ2 (ion channel gene underlying IK1), SCN5A, and MLC2v.

TABLE 1 Characteristics of Nodal/Pacemaker and Atrial/Ventricular(“Working”) Cardiomyocytes N dV/dt_(max) (V/s) APD₅₀ (ms) APA (mV) MDP(mV) Rate (bpm) Nodal-like CMs 14  6.5 ± 4.1** 104.3 ± 26.4* 74.9 ±11.2** −47.2 ± 4.6** 122.0 ± 36.5* Working CMs 31 44.2 ± 31.7 144.7 ±48.9 96.8 ± 14.9 −57.5 ± 8.9  85.8 ± 29.0 Data are mean ± SD.Abbreviations are as follows: N = cell number, APD₅₀ = action potentialduration measured at 50% repolarization, dV/dt_(max) = maximum rate ofaction potential upstroke, APA = action potential amplitude, MDP =maximum diastolic potential. **P < 0.01 and *P < 0.001 nodal-like vs.working CMs.

Another aspect of the present invention is directed to a pharmaceuticalcomposition comprising the nodal/pacemaker cardiomyocytes produced inaccordance with the methods of the present invention. In a preferredembodiment, the nodal/pacemaker cardiomyocytes of the pharmaceuticalcomposition are human nodal/pacemaker cardiomyocytes, and are producedin accordance with the methods of producing nodal/pacemakercardiomyocytes described supra. The pharmaceutical composition furtherincludes a pharmaceutically acceptable carrier, such as an isotonicexcipient prepared under sufficiently sterile conditions for humanadministration. For general principles in medicinal formulation see CELLTHERAPY: STEM CELL TRANSPLANTATION, GENE THERAPY, AND CELLULARIMMUNOTHERAPY (George Morstyn & William Sheridan eds., CambridgeUniversity Press, 1996) and EDWARD D. BALL et al., HEMATOPOIETIC STEMCELL THERAPY (Churchill Livingstone, 2000).

Choice of the cellular excipient and any accompanying elements of thecomposition will be adapted in accordance with the route and device usedfor administration. The composition may also comprise or be accompaniedwith one or more other ingredients that facilitate the engraftment orfunctional mobilization of the cardiomyocytes upon implantation.Suitable ingredients include matrix proteins that support or promoteadhesion of the cardiomyocytes, or complementary cell types, especiallyendothelial cells. Additional ingredients that may be included topromote survival of cardiomyocytes upon deliver include Matrigel, aBcl-XL peptide, cyclosporine A, a compound that opens ATP-dependent K+channels, IGF-1, and a caspase inhibitor.

Another aspect of the present invention is directed to a method oftreating cardiac arrhythmia in a subject. This method involves providingan isolated population of nodal/pacemaker cardiomyocytes of the presentinvention and delivering the isolated population of nodal/pacemakercardiomyocytes to the subject under conditions effective to treat thecardiac arrhythmia.

In a preferred embodiment of this aspect of the present invention, asubject having a cardiac arrhythmia is selected and the isolatedpopulation of nodal/pacemaker cardiomyocytes is delivered to thisselected subject. The selected subject may have any type of cardiacarrhythmia condition, including, but not limited to, sinus nodedysfunction (e.g. “sick sinus” syndrome), bifascicular block,trifascicular block, third-degree atrial-ventricular block, Stokes-Adamattack, or atrial fibrillation. The selected subject can be any animal,preferably a mammal, having a cardiac arrhythmia. In a preferredembodiment of the present invention, the selected subject is a humansubject.

Preferably, the isolated population of nodal/pacemaker cardiomyocytesare made in accordance with the methods of the present invention.Appropriate methods and reagents for inducing cardiomyocytedifferentiation and producing cardiomyocytes having a nodal/pacemakerphenotype are described supra.

In accordance with this aspect of the present invention, thecardiomyocytes having a nodal/pacemaker phenotype can be delivered tosubject having the arrhythmia via means known in the art, including acatheter-based or direct intramyocardial injection during surgery. In apreferred embodiment of the present invention, the nodal/pacemaker cellsare delivered by catheter guided by simultaneous or previouselectroanatomic mapping procedures (e.g., the NOGA system) which areused to diagnose, localize, and treat certain cardiac rhythmdisturbances. These methods are known in the art, see e.g., Perin etal., “Stem Cell Therapy in End-Stage Ischaemic Heart Failure: ACatheter-Based Therapeutic Strategy Targeting Myocardial Viability,”Euro. Heart J. 8(Suppl. H):H46-H51 (2006) and Psaltis et al.,“Intramyocardial Navigation and Mapping for Stem Cell Delivery,” J ofCardiovasc. Trans. Res. DOI 10.1007/s12265-009-9138-1 (2009), which arehereby incorporated by reference in their entirety). In anotherembodiment of the present invention, magnetic resonance (MR) guidedintramyocardial delivery of the nodal/pacemaker cardiomyocytes, asdescribed by Karmarkar et al., “MR-Trackable Intramyocardial InjectionCatheter,” Magnetic Resonance in Medicine 51(6):1163-72 (2004), which ishereby incorporated by reference in its entirety, is used for deliveringcells to the desired location in the heart (i.e., the native SA node orAV node structures or elsewhere in the conduction system).

While a catheter-based approach for delivering the pacemaker cells to asubject are superior, these cells can also be delivered directly byintramuscular injection via thoracotomy (e.g., by the surgeon duringcoronary bypass grafting). Other alternatives to catheter-based deliveryinclude intracoronary infusion, intravenous injection, bolus injectionvia a catheter during a surgical procedure such as a percutaneoustransluminal coronary angioplasty, transendocardial injection,transvascular injection, intramuscular injection, or intra-arterialinjection.

One or more injections, infusions, or implantations of cardiomyocytesmay be necessary to provide pacemaker activity in larger mammals such ashumans. Anticipated total graft sizes in humans are from about 2.5×10⁴to 1×10⁸ cells or more. These established, viable grafted cells can beprovided by one or multiple cellular implantations, e.g., by implantingup to about 10⁸ or more cells at a time. In a preferred embodimentcardiomyocytes are delivered to the target heart tissue multiple times,delivering up to about 2.5×10⁴ to 1×10⁸ cells at a time to add to thesize of the graft and optimize its biological pacing or other activity.

The cardiomyocytes of the present invention are administered in a mannerthat permits them to graft or migrate to the intended tissue site andreconstitute or regenerate the functionally deficient area. To reducethe risk of cell death upon engraftment, the cells may be treated byheat shock or cultured with 0.5 U/mL erythropoietin 24 hours beforeadministration. Where desirable, the patient receiving an allograft ofcardiomyocytes can be treated to reduce immune rejection of thetransplanted cells. Methods contemplated include the administration oftraditional immunosuppressive drugs like cyclosporin A (Dunn et al.,“Cyclosporin: An Updated Review of the Pharmacokinetic Properties,Clinical Efficacy and Tolerability of a Microemulsion-Based Formulation(neoral)1 in Organ Transplantation,” Drugs 61:1957 (2001), which ishereby incorporated by reference in its entirety), or inducingimmunotolerance (see e.g., WO2002/44343 to Chiu et al.; WO2003/050251 toBhatia et al.; and U.S. Pat. No. 6,280,718 to Kaufman, which are herebyincorporated by reference in their entirety). Another approach is toadapt the cardiomyocyte cell population to decrease the amount of uricacid produced by the cells upon transplantation into a subject, forexample, by treating them with allopurinol. Alternatively or inconjunction, the individual is prepared by administering allopurinol, oran enzyme that metabolizes uric acid, such as urate oxidase (see e.g.,WO2005/06630 to Gold, which is hereby incorporated by reference in itsentirety).

Another aspect of the present invention is directed to a method ofproducing cardiomyocytes having an atrial/ventricular phenotype. Thismethod involves culturing stem cells under conditions effective toproduce cardiomyocytes and contacting the cardiomyocytes with aneuregulin-1 agonist, neuregulin-1 mimetic, or a related agonist of anErbB receptor under conditions effective to induce the production ofcardiomyocytes having an atrial/ventricular phenotype.

Cardiomyocytes having an atrial/ventricular phenotype, also referred toas a “working” phenotype are the reciprocal of nodal cardiomyocytes. Theunique electrophysiological and molecular characteristics ofatrial/ventricular cells produced in accordance with the methods of thepresent invention are described in more detail below and are listed inTable 1 above.

Appropriate stem cells and reagents for producing differentiatedcardiomyocytes suitable for use in this aspect of the invention aredescribed supra.

In accordance with this aspect of the invention, production ofcardiomyocytes having an atrial/ventricular phenotype involves culturingcardiomyocytes with a neuregulin-1 agonist, neuregulin-1 mimetics, orrelated agonists of the corresponding ErbB receptors (ErbB2, ErbB3, andErbB4). Suitable agonists of both neuregulin-1 and ErbB includerecombinant protein or peptide fragments of neuregulin-1 or other ErbBreceptor ligands and the nucleic acid molecules encoding the same,antibody agonists, and small molecule agonists.

Recombinant protein or peptide fragments of neuregulin suitable for usein the present invention include any neuregulin protein or peptide thatcan bind to and activate ErbB2, ErbB3, ErbB4 or combinations thereof,including but not limited to any of the neuregulin isoforms (Genbank andprotein accession numbers for neuregulin-1 are described supra),polypeptides comprising the neuregulin epidermal growth factor-like(EGF) domain alone, polypeptides containing the neuregulin EGF-likedomain, neuregulin mutants or derivatives, and any kind ofneuregulin-like gene product that encodes the aforementioned protein andpeptide fragments that activate the ErbB receptors. In a preferredembodiment, the neuregulin protein or peptide fragments of the presentinvention are human neuregulin-1β protein or peptide fragments.

As discussed supra, recombinant neuregulin peptides consisting of theEGF-like domain alone are sufficient to activate the appropriate ErbBreceptors. As used herein, “EGF-like domain” refers to a polypeptidemotif encoded by the neuregulin gene that binds to and activates ErbB2,ErbB3, ErbB4, or combinations thereof, and bears a structural similarityto the epidermal growth factor receptor-binding domain (see e.g.,Buonanno & Fischbach, “Neuregulin and ErbB Receptor Signaling Pathwaysin the Nervous System,” Curr Opin Neurobiol 11:287-96 (2001) (showingalignment of the human EGF-like domains in neuregulins 1-4 and other EGFrelated ligands), which is hereby incorporated by reference in itsentirety). Exemplary recombinant peptides containing the neuregulinEGF-like domain that are known to activate ErbB signaling are disclosedin WO2000/64400 to Marchionni et al.; WO1997/09425 to Chang;WO2000/037095 to Zhou et al.; and WO2003/020911 to Stefansson et al.,which are hereby incorporated by reference in their entirety.

Suitable ErbB agonists for use in accordance with this aspect of thepresent invention include the recombinant neuregulin protein and peptidefragments discussed supra. In addition, ErbB receptor ligands or ligandfragments other than neuregulin that bind to and activate ErbB includingheregulin, amphiregulin, betacellulin, and epiregulin, are also suitablefor purposes of the present invention. Recombinant heregulin,amphiregulin, betacellulin, and epiregulin ErbB receptor ligands, oractive peptide fragments thereof, are disclosed in U.S. PatentPublication Nos. 20010007657 to Reid et al.; 20080031880 to Huang etal.; 20070054851 to Lin et al.; and U.S. Pat. No. 6,136,558 to Ballingeret al., which are all hereby incorporated by reference in theirentirety.

In another embodiment of the present invention, the agonist of ErbB isan ErbB receptor agonist antibody (see e.g., U.S. Patent Publication No.20080031880 to Huang et al., and Amin et al., “Gene Expression Profilingof ErbB Receptor and Ligand-Dependent Transcription,” Oncogene23:1428-38 (2004), which are hereby incorporated by reference in theirentirety).

Another aspect of the present invention is directed to an isolatedpopulation of atrial/ventricular cardiomyocytes. In a preferredembodiment, the isolated population of atrial/ventricular cardiomyocytesare human atrial/ventricular cardiomyocytes produced in accordance withthe methods of the present invention described herein. The isolatedpopulation of human atrial/ventricular cardiomyocytes of the presentinvention are characterized by electrophysiological characteristicsincluding a slower spontaneous firing rate (mean of ≅85 bpm), a veryrapid action potential upstroke (>>15 V/s, mean of 45 V/s), a longeraction potential duration, and substantial fast sodium current (SeeTable 1, above). Correspondingly, these cells show strong expression ofthe ion channel genes SCN5A and KCNJ2 and reduced expression of the HCNpacemaker family genes. The atrial/ventricular cells of the presentinvention express usual pan-cardiac markers (sarcomeric actin,sarcomeric myosin, troponins) and strongly expressworking/chamber-specific markers appropriate for developmental stage(e.g., MLC2v, atrial natriuretic factor). These cells retain thesephenotype markers with maturation; moreover, unlike pacemaker cells,working cardiomyocytes should hypertrophy and show increased single-cellforce generation with development.

Another aspect of the present invention is directed to a pharmaceuticalcomposition containing the atrial/ventricular cardiomyocytes of thepresent invention. In a preferred embodiment, the pharmaceuticalcomposition contains an isolated population of human atrial/ventricularcardiomyocytes. The pharmaceutical composition further contains apharmaceutically acceptable carrier and additional ingredients thatfacilitate engraftment and mobilization upon implantation as describedsupra for the composition containing the nodal/pacemaker cardiomyocytes.In a preferred embodiment, the isolated population of humanatrial/ventricular cardiomyocytes are produced in accordance with themethods described supra.

Another aspect of the present invention is directed to a method ofimproving cardiac tissue repair or cardiac organ function in a subject.This method involves providing an isolated population ofatrial/ventricular cardiomyocytes and delivering the isolated populationof atrial/ventricular cardiomyocytes to the subject under conditionseffective to improve cardiac tissue repair or cardiac organ function.

In a preferred embodiment of the present invention, the isolatedpopulation of atrial/ventricular cardiomyocytes are produced inaccordance with the methods of the present invention. Appropriatemethods and reagents for inducing cardiomyocytes differentiation andproducing cardiomyocytes having an atrial/ventricular phenotypes aredescribed supra.

In accordance with this aspect of the present invention a subject inneed of cardiac tissue repair or improved cardiac organ function isfirst selected and the isolated population of atrial/ventricularcardiomyocytes are delivered to the selected subject. Individuals inneed of cardiac tissue repair or improved cardiac organ function who aresuitable for receiving cardiomyocyte based therapy include thosesuffering from or having suffered from congestive heart failure,myocardial infarction, coronary heart disease, cardiomyopathy,endocarditis, congenital cardiovascular defects, and congestive heartfailure, or any condition resulting in cardiac tissue injury (e.g.,ischemia, apoxia, hypoxia, etc.)

Methods of delivering the cardiomyocytes having atrial/ventricularphenotypes include catheter-based or direct intramyocardial injectionand intracoronary infusion as described supra. In accordance with thisaspect of the present invention, the cardiomyocytes havingatrial/ventricular phenotypes can be delivered as a composition whichincludes a biocompatible scaffold to further facilitate tissueregeneration (See WO2005/095652 to Ebert et al., which is herebyincorporated by reference in its entirety).

In a preferred embodiment of this aspect of the present invention, thecardiomyocytes will be delivered to the region of, or adjacent to theregion of injured myocardium, and will be introduced as soon as possibleafter the infarct or other injury. Preferably, the cardiomyocytes aredelivered during active granulation tissue formation but prior toscarring and myocardial thinning. Preferably, the deliveredcardiomyocyte cells are physically and electronically coupled to theviable native myocardium adjacent to the injured myocardium. Suchcoupling can be observed, for example, by the organization of theengrafted cells and the formation of nascent junctional complexes bothbetween engrafted cells themselves and between engrafted cells and thenative cardiomyocytes. Several implantations of cardiomyocytes may benecessary to provide the restorative tissue properties. These may beprovided, for example, by one or more infusions, implantations, orinfusions of cells, with each delivery having up to about 1×10⁶ to 1×10⁹cells or more. In a preferred method, multiple cellular administrations(e.g., injections) of 2.5×10⁵ to 2.5×10⁶ cells at a time to achieve anappropriate graft size of 1×10⁷ to 1×10⁹ cells. At least oneimplantation of cells will be made such that the engrafted cells contactviable myocardial tissue on the perimeter of the injured region ofmyocardium. Where a larger region of injury is involved, multipleinjections or implantations of cells may be made around the periphery ofthe injured region so as to substantially surround the injured regionwith engrafted, viable cells. Furthermore, in subsequent implantationsconducted in the same operative procedure or in follow-up procedures,cells from a prior engraftment may be used as the newly establishedperiphery, and grafts may be constructed so as to provide viable,coupled cardiomyocytes substantially into or substantially throughoutthe injured myocardial region, so as to provide the improvedfunctionality to the region. After one or multiple procedures forrestorative engraftment, the individual can be monitored for improvementin contractile function of the heart and the loss or decrease infunctional artifacts caused by the injured region of myocardium. Suchmonitoring can be conducted by signal processing methodologies which areknown and used in the detection and localization of ischemic or otherinjured myocardium. The efficacy of cellular delivery treatment can alsobe monitored by the reduction in area occupied by scar tissue orrevascularization of scar tissue, and in the frequency and severity ofangina; or an improvement in developed pressure, systolic pressure, enddiastolic pressure, Δpressure/Δtime, patient mobility, and quality oflife.

The present invention is illustrated, but not limited, by the followingexamples.

EXAMPLES Example 1 Generation and Characterization of hESC-CMs

H7 human embryonic stem cells (H7 hESCs) were maintained in anundifferentiated state on Matrigel (BD Biosciences) coated plates inmouse embryonic fibroblast conditioned medium (MEF-CM) supplemented with4 ng/ml bFGF (Peprotech), as previously described (Xu et al.,“Feeder-Free Growth of Undifferentiated Human Embryonic Stem Cells,”Nat. Biotechnol. 19(10):971-974 (2001), which is hereby incorporated byreference in its entirety). H7 hESCs were induced to differentiate intocardiomyocytes by the sequential application of 100 ng/ml activin for 24hours followed by 4 days of 10 ng/ml BMP4. After 1 week in RPMI/B27without added growth factors, cells were dispersed using Blendzyme 4(Roche) and plated overnight onto glass-bottom Petri dishes, usingmedium containing 20% fetal calf serum (Hyclone). On the following day,the medium was returned to serum-free medium and cells were cultured foran additional 7-27 days. The spontaneously generated action potentialsof hESC-derived cardiomyocytes at 35-36° were then recorded using a HEKAEPC-10 amplifier (which has a true “voltage follower” circuit, similarto a classical microelectrode amplifier), controlled by a Dell OptiplexGX280 SMT Pentium 4 computer and operated in current clamp mode. Afterobtaining gigaohm seal, electrical access to the cells was obtained viathe β-escin perforated patch technique. The capacitance of the examinedcells was 17.5±7.6 pF (range 5.8-32.8 pF), in comparison to the ˜150 pFtypically reported for adult human ventricular myocytes. Bath medium was(in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 0.33 NaH₂PO4, 5dextrose, and 10 HEPES, adjusted to pH 7.40 with NaOH. The pipettesolution was (in mM) 135 KCl, 5 Na₂creatine phosphate, 5 MgATP and 10HEPES, adjusted to pH 7.20 with KOH. Data were digitized at 10 kHz andfiltered at 2 kHz. Action potential parameters were analyzed usingPatchmaster (HEKA) and Igor Pro software.

Example 2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)Analysis

Total RNA was prepared by lysing cell preparations with the QiagenRNEasy kit, followed by DNase treatment. After confirming the quality ofthe RNA using an Agilent Bioanalyzer 2100, it was reverse-transcribedinto cDNA using the Superscript III first-strand cDNA synthesis kit(Invitrogen). Tables 2 and 3 list the primers used for semi-quantitativeand quantitative RT-PCR reactions, respectively. All primer pairs weredesigned to be intron-spanning. Quantitative real-time PCR reactionswere performed using the SYBR green dye system and an Applied Biosystems7900HT instrument. Cycling conditions were 10 minutes at 95° C., and 40cycles of 30 seconds at 95° C., 30 seconds at 55° C., and 30 seconds at72° C. mRNA levels were normalized using GAPDH as an internal control,and adult human heart cDNA was always run in parallel as a positivecontrol.

TABLE 2 Primer Sets Used for Semi-Quantitative RT-PCR Forward PrimerSequence Product Size Annealing Temp Gene Reverse Primer Sequence (bp)(° C.) RG-1α TTGCTCCAGTGAATCCAGGTT 34 55 (SEQ ID NO: 8)TGAAAAGCCAGGAATCGGCTG (SEQ ID NO: 9) RG-1β CGATCACCAGTAAACTCATTTG 35 55(SEQ ID NO: 10) TGAAAAGCCAGGAATCGGCTG (SEQ ID NO: 11) rbB1GGCATAGGAATTTTCGTAGTACATAT 50 60 (SEQ ID NO: 12) GACCCTCCGGGACGG (SEQ IDNO: 13) rbB2 GGGGCTGGGGCAGCCGCTC 31 60 (SEQ ID NO: 14)GGCTGCTGGACATTGACGAG (SEQ ID NO: 15) rbB3 CAGGTCTGGCAAGTATGGAT 27 60(SEQ ID NO: 16) GGAGTACAAATTGCCAAGGGTA (SEQ ID NO: 17) rbB4CATTGTATTCTTTTTCATCTCCTTC 24 60 (SEQ ID NO: 18) CTCTGATCATGGCAAGTATGGAT(SEQ ID NO: 19) β-actin CAAGGCCAACCGCGAGAAGATGAC 20 58 (SEQ ID NO: 20)AGGAAGGAAGGCTGGAAGAGTGC (SEQ ID NO: 21)

TABLE 3 Primer Sets Used for Quantitative RT-PCR Analysis AliasesForward Primer Sequence Gene (Associated Currents) Accession ID ReversePrimer Sequence CACNA1C Cav1.2 (I_(CaL)) NM 000719 CAGAGGCTACGATTTGAGGA(SEQ ID NO: 22) GCTTCACAAAGAGGTCGTGT (SEQ ID NO: 23) CANCA1G Cav3.1(I_(CaT)) NM 198397 CTTCACACCATATGCTGTCT (SEQ ID NO: 24)CTGCTCCACCATGTAGCTCT (SEQ ID NO: 25) GJA1 Cx43 NM 000165CTTTTGGAGTGACCAGCAAC (SEQ ID NO: 26) TGAAGCTGAACATGACCGTA (SEQ ID NO:27) GJA5 Cx40 NM 181703 GCAGCCTCAGCTTTACAAATG (SEQ ID NO: 28)GTGACAGATGTTGGCAGGAAT (SEQ ID NO: 29) GJA7 Cx45 NM 005497TCTCACTCGCATCAGAATCA (SEQ ID NO: 30) AAGAGCAAAGGACACACCAC (SEQ ID NO:31) HCN1 (I_(f)) NM 021072 GTGACAGAAAGCAGGGGTAA (SEQ ID NO: 32)ATTGCCAGTGCCAGAGATAC (SEQ ID NO: 33) HCN2 (I_(f)) NM 001194AGCTCAAGTTCGAGGTCTTCC (SEQ ID NO: 34) TCTCCTTGTTGCCCTTAGTGA (SEQ ID NO:35) HCN4 (I_(f)) NM 005477 TGATGGTGGGAAACCTGATTA (SEQ ID NO: 36)GTTGAGGACCAAGTCGATGAG (SEQ ID NO: 37) KCNE1 mink, ISK NM 000219CAGGCCAGATTTACAGGAGA (I_(Ks)) (SEQ ID NO: 38) GCAGAATCAGTGTGTGCTTG (SEQID NO: 39) KCNE2 MiRP1 NM 172201 TCATGGTGATGATTGGAATG (I_(Kr), I_(f),I_(to)) (SEQ ID NO: 40) TTATCAGGGGGACATTTTGA (SEQ ID NO: 41) KCNJ2Kir2.1 (I_(K1)) NM 000891 TTGTCAAGAGCCAAGACACA (SEQ ID NO: 42)AGCAACACACATCTGGGAAT (SEQ ID NO: 43) MLC-2a AK311869TCAGCTGTATCGACCAGAATCG (SEQ ID NO: 44) AAGACGGTGAAGTTGATGGG (SEQ ID NO:45) MLC-2v NM 000432 CGTTCGGGAAATGCTGACCACGC (SEQ ID NO: 46)AGTCCAAGTTTCCAGTCACGTCAG (SEQ ID NO: 47) NKX2-5 NM 004387CCCTGGATTTTGCATTCACT (SEQ ID NO: 48) GGGGACAGCTAAGACACCAG (SEQ ID NO:49) NPPA ANF NM 006172 ACAGACGTAGGCCAAGAGAG (SEQ ID NO: 50)GTCTGACCTAGGAGCTGGAA (SEQ ID NO: 51) SCN5A Nav1.5 (I_(Na)) NM 000335AGCTCTGTCACGATTTGAGG (SEQ ID NO: 52) AGGACTCACACTGGCTCTTG (SEQ ID NO:53) TBX2 NM 005994 CTGGACAAGAAGGCCAAGTA (SEQ ID NO: 54)GCATGGAGTTTAGGATGGTG (SEQ ID NO: 55) TBX3 NM 016569 ATTTCACAATTCTCGGTGGA(SEQ ID NO: 56) TATAATTCCCCTGCCACGTA (SEQ ID NO: 57) TBX5 NM 080718TCCAGAAACTCAAGCTCACC (SEQ ID NO: 58) TGGCAAAGGGATTATTCTCA (SEQ ID NO:59)

Example 3 Western Blot Analysis of Akt and Erk Phosphorylation

To demonstrate functional NRG1/ErbB signaling in hESC-CM cultures, twoeffectors in this signal transduction cascade, the Akt and Erk kinases,were analyzed by western blotting. For this, hESC-CMs (˜5×10⁶ millionper well) were treated on day 10 post-induction with 0, 10, or 100 ng/mlNRG-1β (R&D Systems) in the presence or absence of 25 μg/ml anti-NRG1βneutralizing antibody (R&D Systems) for 10 minutes. After treatment,cells were rinsed twice with ice-cold PBS and lysed for 20 minutes onice with 250 μl of extraction buffer (25 mM Tris-HCl (pH 7.4), 150 mMNaCl, 2 mM EDTA, 10 mM Na₂SO₄, and 1% Triton X-100 supplemented justprior to use with 50 mM NaF, 1 mM sodium orthovanadate, 1 mMphenylmethylsulfonyl fluoride, and a protease inhibitor cocktail(Sigma)). Lysates were collected with a cell scraper, placed inmicrocentrifuge tubes, vortexed for 3 minutes at 4° C., and then spun at4° C. for 20 minutes at 12,000 g to discard cell debris. The resultantprotein lysates were then denatured at 95° C. for 10 minutes and loaded(at 30 μg/lane) onto a denaturing 10% SDS/polyacrylamide gel,electrophoresed, and then transferred onto a PVDF membrane. The PVDFblot was blocked for 1 hour at room temperature in 1×TBST (150 mM NaCl,10 mM Tris-HCl (pH 7.4), and 0.1% Tween) plus 5% nonfat milk, probedovernight with anti-phosphorylation Akt and anti-phosphorylation Erk1/2antibodies (Cell Signaling), and then incubated withperoxidase-conjugated goat anti-mouse IgG antibody (Sigma). Theresultant bands were visualized using an enhanced chemiluminescentdetection kit (Amersham). The membrane was then stripped and exposed tototal Akt and Erk antibodies (Cell Signaling) to detect total Akt andErk.

Example 4 Current-Clamp Studies

At 14 days following induction with activin A, hESC-CMs were dispersedusing 0.1 U/ml of dispase (Invitrogen) with 63 U/ml DNase I (Invitrogen)and replated at low density onto 0.5% gelatin-coated glass coverslips.After a few days of additional culture, the spontaneously generatedaction potentials (APs) of the hESC-CMs were recorded using a HEKAEPC-10 amplifier (HEKA, Lambrecht, Germany), operated in current-clampmode. Note that the EPC-10 has a true “voltage follower” circuit,similar to a classical microelectrode amplifier (Magistretti et al.,“Modalities of Distortion of Physiological Voltage Signals byPatch-Clamp Amplifiers: a Modeling Study,” Biophys J. 74(2 Pt 1):831-842(1998) and Magistretti et al., “Action Potentials Recorded withPatch-Clamp Amplifiers: Are They Genuine?” Trends Neurosci.19(12):530-534 (1996), which are hereby incorporated by reference intheir entirety). After obtaining gigaohm seal, electrical access to thecells was obtained via the β-escin perforated patch technique (Fan etal., “Perforated Patch Recording with Beta-escin,” Pflugers Arch.436(6):1021-1023 (1998) and Fu et al., “Perforated Patch Recording ofL-type Calcium Current with Beta-escin in Guinea Pig VentricularMyocytes,” Acta Pharmacol Sin. 24(11):1094-1098 (2003), which are herebyincorporated by reference in their entirety), which was found to improvethe success relative to the conventional ruptured patch approach, as itgreatly minimized both seal disruption and rundown. Patch pipettes witha resistance of 2-4 MΩ were used; cells with a series resistance of >10MΩ were discarded. The capacitance of the examined cells was 17.5±7.6 pF(range 5.8-32.8 pF), in comparison to the ˜150 pF typically reported foradult human ventricular myocytes (Drouin et al., “ElectrophysiologicCharacteristics of Cells Spanning the Left Ventricular Wall of HumanHeart Evidence for Presence of M Cells,” J Am Coll Cardiol.26(1):185-192 (1995), which is hereby incorporated by reference in itsentirety). All recordings were performed at 36±1° C., using thefollowing bath medium: (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.0 MgCl₂,0.33 NaH₂P0₄, 5 dextrose, and 10 HEPES, adjusted to pH 7.40 with NaOH.The pipette solution was (in mM) 135 KCl, 5 Na₂ creatine phosphate, 5MgATP and 10 HEPES, adjusted to pH 7.20 with KOH. Data were digitized at10 Hz and filtered at 2.9 Hz. Action potential parameters were analyzedby an individual blinded to culture conditions, using Patchmaster (HEKA)and Igor Pro software.

Example 5 Generation and Use of the cGATA6-EGFP Lentiviral Vector

The pPD46.21 plasmid containing the proximal (−1.5/+0.0)promoter-enhancer region of the chicken GATA6 (cGATA6) gene wasgenerously provided by Dr. John Burch (Fox Chase Cancer Center) (Daviset al. “A GATA-6 Gene Heart-Region-Specific Enhancer Provides a NovelMeans to Mark and Probe a Discrete Component of the Mouse CardiacConduction System,” Mech Dev. 108(1-2):105-119 (2001), which is herebyincorporated by reference in its entirety). To generate the cGATA6-EGFPlentiviral vector, the 1.5 kb cGATA6 promoter-enhancer region wasexcised from the pPD46.21 plasmid by digestion with the restrictionenzymes Sal I and Age I. This promoter-enhancer fragment was ligatedinto the lentiviral transfer plasmid pJGL2-EGFP (generously provided byDrs. Jonathan Golob and Charles Murry, University of Washington), whichincludes a transgene in which EGFP expression is driven by theconstitutive elongation factor-1α (EF1α) promoter, a central polypurinetract, and a woodchuck hepatitis virus post-transcriptional regulatoryelement (Barry et al., “Lentivirus Vectors Encoding Both CentralPolypurine Tract and Posttranscriptional Regulatory Element ProvideEnhanced Transduction and Transgene Expression,” Hum Gene Ther.12(9):1103-1108 (2001), which is hereby incorporated by reference in itsentirety). For this, the EF1α promoter DNA was excised from pJGL2-eGFPplasmid, also using Sal I and Age I digestion, followed by replacementby the cGATA6 promoter-enhancer fragment.

VSV-G-pseudotyped lentiviral particles were generated and concentratedas previously described (Barry et al., “Lentivirus Vectors Encoding BothCentral Polypurine Tract and Posttranscriptional Regulatory ElementProvide Enhanced Transduction and Transgene Expression,” Hum Gene Ther.12(9):1103-1108 (2001) and Li et al., “Stable Transduction of MyogenicCells with Lentiviral Vectors Expressing a Minidystrophin,” Gene Ther.12(14):1099-1108 (2005), which are hereby incorporated by reference intheir entirety). In brief, 6×10⁶ HEK293D cells seeded on a 15 cm² plate24 hours prior to co-transfection with the following plasmids: 8 μg ofenvelope plasmid pMK-VSVG, 15 μg of pMDL-G/P-RPE plasmid expressing theHIV-1 gap/pol and tat genes, 11.5 μg of pRSV-REV plasmid expressing theHIV-1 rev protein, and 29 μg of either the cGATA6-EGFP or EF1α-EGFPlentiviral transfer vector construct. Supernatant containing theresultant viral particles was collected at 72 hours followingtransfection, concentrated by filtration (Millipore Centricon Plus-20columns with a molecular weight cutoff of 10 kD), and stored at −80° C.Lentiviral stocks were titered by viral p24^(gag) ELISA (QuickTiterLentiviral Quantitation kit, Cell Biolabs).

Prior to lentiviral transduction, hESC-CMs were replated onto glasscoverslips as described for electrophysiological studies and allowed torecover for 4-5 days. Cells were then exposed to cGATA6-EGFP lentivirus(at 5000 LPs/cell, added to their usual RPMI-B27 medium) for 12 hours.Parallel transduction with an equivalent quantity of theconstitutively-expressing EF1α-EGFP lentiviral vector was routinelyperformed and indicated that this viral titer results in the reliabletransduction of ˜50% of target hESC-CMs. Transduced cell preparationswere then used in either electrophysiological or immunocytochemicalstudies at 3-4 days post-transduction.

Example 6 Immunocytochemistry

Dissociated hESC-CMs were cultured on glass cover slips, fixed, andimmunostained as previously described (Laflamme et al., “CardiomyocytesDerived from Human Embryonic Stem Cells in Pro-Survival Factors EnhanceFunction of Infarcted Rat Hearts,” Nat Biotechnol. 25(9):1015-1024(2007), McDevitt et al., “Proliferation of Cardiomyocytes Derived fromHuman Embryonic Stem Cells is Mediated Via the IGF/PI 3-Kinase/AktSignaling Pathway,” J Mol Cell Cardiol. 39(6):865-873 (2005), Laflammeet al., “Formation of Human Myocardium in the Rat Heart from HumanEmbryonic Stem Cells,” Am J Pathol. 167(3):663-671 (2005), and Minami etal., “Extracardiac Progenitor Cells Repopulate Most Major Cell Types inthe Transplanted Human Heart,” Circulation 112(19):2951-2958 (2005),which are hereby incorporated by reference in their entirety).Immunocytochemistry was performed with primary antibodies directedagainst α-sarcomeric actin (clone 5C5, Sigma, 1:2500 titer), troponin Tcardiac isoform Ab-1 (clone 13-11, Thermo Scientific, 1:100), HCN4(clone N114/10, UC Davis/NIH NeuroMab facility, 1:100), and EGFP (NovusBiologicals, 1:1000), and the β-myosin heavy chain isoform (cloneA4.951, American Type Culture Collection, 1:10). Unless otherwisestated, EGFP expression was confirmed in all studies using theaforementioned anti-EGFP antibody. Detection was performed usingAlexa-488 or -594 conjugated secondary antibodies (Molecular Probes).All cell counts were performed by an observer blinded as to precedingtreatment conditions. Each experimental condition was assayed intriplicate, and a minimum of 500 nuclei were counted per sample.

Example 7 Statistics

When analyzing the proportion of hESC-CMs exhibiting each actionpotential phenotype under various treatment conditions, groups werecompared using Fisher's exact test with Bonferroni correction (withα=0.05 for significance). In the statistical analysis of all otherexperiments, ANOVA followed by post hoc Student's t-testing withBonferroni correction was used. Values are expressed as means±standarderror, unless otherwise stated.

Example 8 hESC-CMs Include Cardiomyocytes with Distinct Nodal andWorking (Chamber)-Type Action Potential Phenotypes

The spontaneous action potential (AP) properties of hESC-CMs resultingfrom the standard directed cardiac differentiation protocol (Laflamme etal., “Cardiomyocytes Derived from Human Embryonic Stem Cells inPro-Survival Factors Enhance Function of Infarcted Rat Hearts,” NatBiotechnol. 25(9):1015-1024 (2007), which is hereby incorporated byreference in its entirety) were characterized. Previous studies havereported that hESC-CMs exhibit distinct either nodal or working-type APphenotypes (He et al., “Human Embryonic Stem Cells Develop into MultipleTypes of Cardiac Myocytes: Action Potential Characterization,” Circ Res.93(1):32-39 (2003), Mummery et al., “Differentiation of Human EmbryonicStem Cells to Cardiomyocytes: Role of Co-culture with VisceralEndoderm-Like Cells,” Circulation 107(21):2733-2740 (2003), and Moore etal., “Distinct Cardiogenic Preferences of Two Human Embryonic Stem Cell(hESC) Lines are Imprinted in Their Proteomes in the Pluripotent State,”Biochem Biophys Res Commun. 372(4):553-558 (2008), which are herebyincorporated by reference in their entirety), but these studies havetypically examined cardiomyocytes generated by microdissectingspontaneously beating foci from embryoid body-derived preparations ofrelatively low cardiac purity. While the latter approach couldpotentially favor cells with greater automaticity, the greater cardiacpurity of the preparations described herein allowed the analysis of allcells in an unbiased fashion, rather than focusing on cells with greaterspontaneity or a particular morphology. In this initial survey,current-clamp techniques were used to record spontaneous APs from atotal of 49 cells, of which only four showed AP characteristics deemedinconsistent with cardiomyocytes (i.e., an AP duration to 90%repolarization (APD₉₀) of <20 ms). The remaining 45 cells all showedtypical cardiac-type APs with distinct nodal- or working-typecharacteristics (FIGS. 1A and 1B).

To establish objective criteria for this classification, histogram plotsfor a variety of parameters including spontaneous firing rate, APD,upstroke velocity (dV/dt_(max)), AP amplitude (APA), and maximaldiastolic potential (MDP) were analyzed. In this analysis, there was aclear-cut bimodal distribution with regard to dV/dt_(max), with a cutoffbetween the two populations of ˜15 V/s. Table 1 summarizes the APparameters for the two populations defined using this threshold (i.e.,defining cells with a dV/dt_(max)<15 V/s as nodal, and those withdV/dt_(max)>15 V/s as working-type). Using this criterion, 31% (14 of 45cells) were classified as nodal-type, with these cells showing asubstantially greater spontaneous firing frequency, a smaller APA, and amore depolarized MDP than did the majority population of working-typecardiomyocytes. Although other investigators have further stratifiedESC-CMs into atrial, ventricular, and even Purkinje fibercardiomyocytes, the electrophysiological distinctions between some ofthese subtypes are known to be subtle at this state of maturation andwere not obvious when analyzing the AP data. Therefore, it was decidedto focus on the unambiguous electrophysiological differences betweennodal and working-type hESC-CMs.

Example 9 Activation of the cGATA6-EGFP Transgene Identifies hESC-CMswith the Nodal Phenotype

While AP phenotyping under current-clamp is considered the“gold-standard” method of determining cardiac subtype, a higherthroughput, molecular approach to phenotyping was developed. Since novalidated markers for early human nodal cells were available, thehypothesis that the activation of a proximal promoter-enhancer elementfrom the chicken GATA6 (cGATA6) gene would specifically identifynodal-type hESC-CMs was tested. In a series of elegant fate-mappingstudies in transgenic mice, it has been demonstrated that this promoterelement is selectively activated in the atrioventricular (AV) node andthe bundle of His of the adult heart (Davis et al., “A GATA-6 GeneHeart-Region-Specific Enhancer Provides a Novel Means to Mark and Probea Discrete Component of the Mouse Cardiac Conduction System,” Mech Dev.108(1-2):105-119 (2001), which is hereby incorporated by reference inits entirety). Moreover, the cGATA6 promoter is active very early incardiac development, showing preferential activity in regions of thecardiac crescent and heart tube fated to contribute to nodal structures(Davis et al., “A GATA-6 Gene Heart-Region-Specific Enhancer Provides aNovel Means to Mark and Probe a Discrete Component of the Mouse CardiacConduction System,” Mech Dev. 108(1-2):105-119 (2001), which is herebyincorporated by reference in its entirety), as well as in nodal cellsderived from murine ESCs (White et al., “Embryonic Stem Cells Form anOrganized, Functional Cardiac Conduction System In Vitro,” Am J PhysiolHeart Circ Physiol. (2004), which is hereby incorporated by reference inits entirety).

To test its function in hESC-CMs, the latter cultures were transducedwith a lentiviral vector in which the proximal cGATA6 promoter drivesexpression of EGFP. Approximately 15% of the resultant cells were EGFP+,and all of the EGFP+ cells immunostained positively for cardiac markerssuch as cardiac troponin T (FIG. 2A), sarcomeric actins, and β-myosinheavy chain (β-MHC) (FIGS. 6A and 6B). The EGFP⁺ cells also uniformlyexpressed the hyperpolarization-activated, pacemaking ion channel geneHCN4 (FIG. 2B), which is perhaps the best validated and earliestexpressed nodal cell marker (Garcia-Frigola et al., “Expression of theHyperpolarization-Activated Cyclic Nucleotide-Gated Cation Channel HCN4During Mouse Heart Development,” Gene Expr Patterns. 3(6):777-783 (2003)and Shi et al., “Distribution and Prevalence ofHyperpolarization-Activated Cation Channel (HCN) mRNA Expression inCardiac Tissues,” Circ Res. 85(1):e1-6 (1999), which are herebyincorporated by reference in their entirety). Next, theelectrophysiological phenotype of the EGFP⁺ and EGFP⁻ cells wascompared. Consistent with the hypothesis that activation of thecGATA6-EGFP transgene would preferentially identify nodal cells, 95% (20of 21) of EGFP+ cells showed a nodal-type AP phenotype, versus only 10%(2 of 20) of the EGFP− cells (FIGS. 2C and 2D). Note that only ˜50% ofhESC-CMs were transduced by the lentiviral vector in these experiments.It is therefore possible that some or all of the small number of EGFP−myocytes with the nodal phenotype actually represent non-transfectedcells. Furthermore, if one corrects for this transduction efficiency,the proportion of cGATA6-EGFP+ putative nodal cells under each conditionis in good agreement with that estimated by AP phenotyping.

Example 10 hESC-CMs Exhibit Intact NRG-1β/ErbB Signaling

Next, studies to demonstrate a functional NRG-1β/ErbB signaling systemin hESC-CM cultures were conducted. RT-PCR analysis confirmed theexpression of NRG-1 agonist as well as ErbB2, ErbB3, and ErbB4 receptorsin both undifferentiated hESC and differentiated hESC-CM cultures (FIG.3A). Time-course studies did not reveal any obvious changes in thelevels of these transcripts in differentiating cultures from day 0through day 30 following the induction of cardiogenesis with activin A.To confirm specific expression of the ErbB receptors by thecardiomyocytes themselves, hESC-CM cultures were dual-immunolabeled withantibodies against ErbB2, ErbB4, and the cardiac marker β-MHC.Consistent with prior findings in rodent hearts (Gassmann et al.,“Aberrant Neural and Cardiac Development in Mice Lacking the ErbB4Neuregulin Receptor,” Nature 378(6555):390-394 (1995), Lee et al.,“Requirement for Neuregulin Receptor erbB2 in Neural and CardiacDevelopment,” Nature 378(6555):394-398 (1995), and Zhao et al.,“Neuregulins Promote Survival and Growth of Cardiac Myocytes.Persistence of ErbB2 and ErbB4 Expression in Neonatal and AdultVentricular Myocytes,”J Biol Chem. 273(17):10261-10269 (1998), which arehereby incorporated by reference in their entirety), ErbB2 and ErbB4were expressed by essentially all of β-MHC⁺ cardiomyocytes, but only bya small minority (<10%) of the β-MHC− non-cardiac cells (FIGS. 3B and3C). Finally, functional NRG1/ErbB signaling in hESC-CM cultures wasdemonstrated by analyzing two proximal effectors in this signaltransduction cascade, the Akt and ERK kinases. Both kinases werephosphorylated in response to treatment with NRG1β ligand but thisresponse was inhibited in the presence of an anti-NRG1β neutralizingantibody (FIG. 3D).

Example 11 Inhibition of NRG-1β/ErbB Signaling Enhances the Proportionof hESC-CMs with the Nodal Phenotype

To test the hypothesis that the NRG-1β/ErbB signaling system regulatesthe relative abundance of the two cardiac subtypes in differentiatinghESC-CM cultures, cardiac differentiation was induced in the presence ofeither inhibitors or activators of NRG-1β/ErbB signaling (FIG. 4A.) TheAP phenotype of the resultant cells under each condition was thendetermined by a blinded electrophysiologist, using the criteriadescribed above. As illustrated in FIG. 4B, the inhibition of NRG1/ErbBsignaling either with anti-NRG1β neutralizing antibody or the ErbBantagonist AG1478 increased the proportion of cells exhibiting anodal-like AP phenotype by nearly three-fold: from 21% in control cellsto 58% and 52% in anti-NRG1β- and AG1478-treated cells, respectively(p<0.05 versus control in both cases). Conversely, there was a trendtoward the opposite effect (i.e., a reduction in the fraction of nodalcells) following treatment with exogenous NRG1β ligand, although thisdid not reach statistical significance after correction for multiplecomparisons.

Similar results were obtained using transgenic hESC-CM cultures in whichthe fraction of nodal-type cells was evaluated by activation of thecGATA6-EGFP transgene (FIG. 4C). Here, the percentage of EGFP+ putativenodal cells increased from 15% in control cultures to 29% in anti-NRG1β-and 25% AG1478-treated cultures (p<0.01 and p<0.05 versus control,respectively). If one again corrects for the ˜50% transductionefficiency, the fraction of nodal-type cells estimated by the geneticlabel under each of these conditions is in reasonable agreement withthat obtained by direct AP recordings.

Example 12 NRG-1β/ErbB Signaling Regulates the Expression of CardiacSubtype-Specific Genes

In the preceding experiments, manipulation of NRG-1β/ErbB signalingchanged the relative abundance of the two cardiac subtypes, asdetermined by both AP phenotyping and activation of the nodal-specificcGATA6-EGFP genetic reporter. To confirm these findings with anindependent, molecular approach, quantitative RT-PCR was used to comparethe expression of 18 subtype-specific genes in control, AG1478, andNRG-1β ligand-treated hESC-CM cultures (see Table 4). Note that radicalchanges in gene expression between these conditions were not expected,as all of the treatment conditions had resulted in a mixture of bothcardiac subtypes by AP phenotyping. Nonetheless, the changes in geneexpression were remarkably consistent with the previous observations: 8of the 18 transcripts evaluated were found to be differentiallyexpressed between treatment conditions, and all 8 transcripts shifted inthe hypothesized directions (FIG. 5). For example, AG1478-treatedhESC-CMs showed 2.6-fold greater expression of the nodal-associatedtranscription factor Tbx-3 (TBX3) (Horsthuis et al., “Gene ExpressionProfiling of the Forming Atrioventricular Node Using a Novel tbx3-BasedNode-Specific Transgenic Reporter,” Circ Res. 105(1):61-69 (2009), whichis hereby incorporated by reference in its entirety) than control cells(p<0.01) and 2.0-fold greater expression of HCN4 (p<0.05). Conversely,hESC-CMs treated with exogenous NRG1β showed 2.6-fold greater expressionof NPPA (i.e., atrial natriuretic factor, ANF) than controls (p<0.05).ANF is a well-validated marker of early working chamber differentiation(Houweling et al., “Developmental Pattern of ANF Gene Expression Revealsa Strict Localization of Cardiac Chamber Formation in Chicken,” AnatRec. 266(2):93-102 (2002), Houweling et al., “Expression and Regulationof the Atrial Natriuretic Factor Encoding Gene Nppa During Developmentand Disease,” Cardiovasc Res. 67(4):583-593 (2005), and Chuva de SousaLopes et al., “Patterning the Heart, A Template for Human CardiomyocyteDevelopment,” Dev Dyn. 235(7):1994-2002 (2006), which are herebyincorporated by reference in their entirety). In sum, activation ofNRG-1β/ErbB signaling increased the expression of genes associated withworking cardiomyocytes, while its blockade with AG1478 increased theexpression of genes associated with the nodal subtype.

TABLE 4 Quantitative RT-PCR Analysis of Cardiac Subtype-Specific GeneExpression in Control, NRG-, and AG1478-hESC-CMs

Fold-gene expression in NRG-1β-treated hESC-CMs, AG1478-treatedhESC-CMs, and adult human heart, in all cases normalized to expressionto control (untreated) hESC-CMs. The primers used in this study arelisted above in Table 3. Values are mean ± SE (from 4 biologicalreplicates). Expression in hESC-CM groups were compared by Bonferronicorrected one-way ANOVA with *P < 0.05 vs. control, **P < 0.01 vs.control, ^(#)P < 0.05 vs. NRG-treated, and ^(##)P < 0.01 vs.NRG-treated. Genes that showed at least one statistically significantdifference between groups are highlighted and also appear in FIG. 5. Thecolumn to the right indicates the pattern of expression hypothesizedbefore the experiment, based on currently published literature.

Discussion of Examples 1-12

Elegant work in non-human model systems indicates that working- andnodal-type cardiomyocytes can be distinguished at a remarkably earlystage during heart development (Christoffels et al., “Architectural Planfor the Heart: Early Patterning and Delineation of the Chambers and theNodes,” Trends Cardiovasc Med. 14(8):301-307 (2004), which is herebyincorporated by reference in its entirety). Working-type myocytes in thenascent atrial and ventricular chambers exhibit greater proliferativeactivity and more rapid electrical propagation than their nodalcounterparts, and they express chamber-specific markers includinghigh-conductance gap junction proteins (connexins-40 and -43) and ANF.Nodal cells show more automaticity and retain a phenotype closer to thatof primary myocardium, in part because the transcription factors Tbx2and Tbx3 repress the chamber-specific gene expression program in thesecells (Bakker et al., “Transcription Factor Tbx3 is Required for theSpecification of the Atrioventricular Conduction System,” Circ Res.102(11):1340-1349 (2008), Christoffels et al., “T-box TranscriptionFactor Tbx2 Represses Differentiation and Formation of the CardiacChambers,” Dev Dyn. 229(4):763-770 (2004), and Hoogaars et al., “TheTranscriptional Repressor Tbx3 Delineates the Developing CentralConduction System of the Heart,” Cardiovasc Res. 62(3):489-499 (2004),which are hereby incorporated by reference in their entirety). Thatsaid, a number of positive markers (including activation of the cGATA6transgene (Davis et al., “A GATA-6 Gene Heart-Region-Specific EnhancerProvides a Novel Means to Mark and Probe a Discrete Component of theMouse Cardiac Conduction System,” Mech Dev. 108(1-2):105-119 (2001) andAdamo et al., “GATA-6 Gene Enhancer Contains Nested Regulatory Modulesfor Primary Myocardium and the Embedded Nascent AtrioventricularConduction System,” Anat Rec A Discov Mol Cell Evol Biol.280(2):1062-1071 (2004), which are hereby incorporated by reference intheir entirety) have been reported to distinguish nodal precursors fromthe remainder of the primary myocardium, and there is increasingevidence for the induction and specialization of nodal cells duringdevelopment (Horsthuis et al., “Gene Expression Profiling of the FormingAtrioventricular Node Using a Novel tbx3-Based Node-Specific TransgenicReporter,” Circ Res. 105(1):61-69 (2009), which is hereby incorporatedby reference in its entirety). He et al., “Human Embryonic Stem CellsDevelop into Multiple Types of Cardiac Myocytes: Action PotentialCharacterization,” Circ Res. 93(1):32-39 (2003), which is herebyincorporated by reference in its entirety, and others have reported thatnodal- and working-type cells can also be distinguished in early hESC-CMcultures using electrophysiological techniques (Mummery et al.,“Differentiation of Human Embryonic Stem Cells to Cardiomyocytes: Roleof Coculture with Visceral Endoderm-Like Cells,” Circulation107(21):2733-2740 (2003) and Moore et al., “Distinct CardiogenicPreferences of Two Human Embryonic Stem Cell (hESC) Lines are Imprintedin Their Proteomes in the Pluripotent State,” Biochem Biophys ResCommun. 372(4):553-558 (2008), which are hereby incorporated byreference in their entirety). Here, these findings have been confirmedand extended by demonstrating that the AP phenotype of hESC-CMscorrelates with the activation of the novel cGATA6 transgene. The aboveExamples also represent the first electrophysiological analysis of thehESC-CMs that result from the recently reported directed differentiationsystem (Laflamme et al., “Cardiomyocytes Derived from Human EmbryonicStem Cells in Pro-Survival Factors Enhance Function of Infarcted RatHearts,” Nat Biotechnol. 25(9):1015-1024 (2007), which is herebyincorporated by reference in its entirety).

This cell culture system was also used to investigate the regulation byNRG-1β/ErbB signaling of cardiac subtype specification indifferentiating human cardiomyocytes. Using three independent approaches(AP phenotyping, activation of the cGATA6-EGFP reporter, and RT-PCRanalysis of subtype-specific genes), it was demonstrated that theinhibition of this signaling pathway results in a ˜2-3 fold increase inthe proportion of hESC-CMs exhibiting the nodal phenotype. Note therewas a trend toward the opposite effect based on AP phenotyping (i.e., adecrease in the fraction of nodal cells following treatment exogenousNRG-1β). Given the findings of abundant endogenous NRG-1β transcript inhESC-CM cultures throughout the differentiation process, it can beinferred that ErbB activation promotes the differentiation(“ventricularization”) and/or expansion of working-type cardiomyocytes,but that this process may be largely saturated by endogenous agonistunder control conditions. Conversely, when endogenous NRG-1β/ErbBsignaling is antagonized, a majority of the resultant hESC-CMs show thenodal phenotype.

In the developing mouse heart, NRG-1β/ErbB signaling has been implicatedin two anatomically and temporally distinct steps: 1) the maturation andexpansion of the primitive ventricle (Meyer et al., “Multiple EssentialFunctions of Neuregulin in Development,” Nature 378(6555):386-390(1995), Kramer et al., “Neuregulins with an Ig-Like Domain are Essentialfor Mouse Myocardial and Neuronal Development,” Proc Natl Acad Sci USA93(10):4833-4838 (1996), Zhao et al., “Selective Disruption ofNeuregulin-1 Function in Vertebrate Embryos Using Ribozyme-tRNATransgenes,” Development 125(10):1899-1907 (1998), Gassmann et al.,“Aberrant Neural and Cardiac Development in Mice Lacking the ErbB4Neuregulin Receptor,” Nature 378(6555):390-394 (1995), Lee et al.,“Requirement for Neuregulin Receptor erbB2 in Neural and CardiacDevelopment,” Nature 378(6555):394-398 (1995), Corfas et al.,“Differential Expression of ARIA Isoforms in the Rat Brain,” Neuron14(1):103-115 (1995), and Hertig et al., “Synergistic Roles ofNeuregulin-1 and Insulin-Like Growth Factor-I in Activation of thePhosphatidylinositol 3-Kinase Pathway and Cardiac ChamberMorphogenesis,” J Biol Chem. 274(52):37362-37369 (1999), which arehereby incorporated by reference in their entirety), and 2) thesubsequent induction of working-type (specifically ventricular)cardiomyocytes into the peripheral conduction system (Rentschler et al.,“Neuregulin-1 Promotes Formation of the Murine Cardiac ConductionSystem,” Proc Natl Acad Sci USA 99(16):10464-10469 (2002) and Patel etal., “Endothelin-1 and Neuregulin-1 Convert Embryonic Cardiomyocytesinto Cells of the Conduction System in the Mouse,” Dev Dyn. 233(1):20-28(2005), which are hereby incorporated by reference in their entirety).The findings described herein are entirely consistent with the formerprocess, that is, the activation of NRG-1β/ErbB signaling promotes therecruitment of early working-type hESC-CMs. Additional mechanisticstudies are required, but this preliminary data suggests NRG-1β/ErbBsignaling regulates differentiation into the working subtype, ratherthan merely differentially affecting the proliferation or survival ofone subtype or another. It would be interesting to examine whetherNRG-1β treatment of later working-type hESC-CMs induces specificPurkinje fiber differentiation, but there is a lack validated markersfor human Purkinje fibers at this developmental stage.

At first glance, the findings described herein may seem to contradicttwo prior studies in non-human models that suggest NRG-1β treatmentactually induces nodal differentiation. First, based on calcium imagingstudies of zebrafish embryos in which NRG was knocked-down by morpholinoantisense oligonucleotides, Milan et al. concluded that NRG was involvedin the patterning of the slow-conducting nodal tissue of the AV ring(Milan et al., “Notch 1b and Neuregulin are Required for Specificationof Central Cardiac Conduction Tissue,” Development 133(6):1125-1132(2006), which is hereby incorporated by reference in its entirety).However, while conduction velocity in the AV node appeared littlechanged in the NRG-morphant hearts, propagation in the atrial andventricular chambers was profoundly slowed (>5-fold). Hence, the moststriking phenotypic change following ablation of NRG-1β/ErbB signalingwas reduced functional maturation of the rapid-conduction chambermyocardium, an observation consistent with the findings described hereinwith hESC-CM cultures. Subsequently, Ruhparwar et al. reported thatNRG-1β induced a “pacemaker-like” phenotype when applied to murineprimary ventricular cardiomyocytes from the late fetal period (Ruhparwaret al., “Enrichment of cardiac pacemaker-like cells: neuregulin-1 andCyclic AMP Increase I(f)-Current Density and Connexin 40 mRNA Levels inFetal Cardiomyocytes,” Med Biol Eng Comput. 45(2):221-227 (2007), whichis hereby incorporated by reference in its entirety). This apparentdiscrepancy can be attributed to imprecision regarding the distinctionbetween nodal (pacemaker) cells and myocytes of the peripheralconduction system (e.g., Purkinje fibers), cell types with uniquedevelopmental origins and phenotypic features (Cheng et al.,“Development of the Cardiac Conduction System Involves RecruitmentWithin a Multipotent Cardiomyogenic Lineage,” Development126(22):5041-5049 (1999), which is hereby incorporated by reference inits entirety). Cells fated to become Purkinje fibers are recruited fromcommitted working-type cardiomyocytes, not from nodal progenitors. It ispossible that Ruhparwar et al induced Purkinje fiber rather than nodaldifferentiation in their study. In support of this assertion, Ruhparwaremphasized that NRG-1β induced a robust increase in connexin-40expression. Connexin-40 is a well-accepted early marker of cardiacchamber differentiation (Van Kempen et al., “Developmental Changes ofConnexin40 and Connexin43 mRNA Distribution Patterns in the Rat Heart,”Cardiovasc Res. 32(5):886-900 (1996), Delorme et al., “DevelopmentalRegulation of Connexin 40 Gene Expression in Mouse Heart Correlates withthe Differentiation of the Conduction System,” Dev Dyn. 204(4):358-371(1995), and Christoffels et al., “Chamber Formation and Morphogenesis inthe Developing Mammalian Heart,” Dev. Biol. 223(2):266-278 (2000), whichare hereby incorporated by reference in their entirety), so an increasein its expression would imply induction of working-type myocytes, not“pacemaker-like” cells as interpreted by the authors. However, becauseconnexin-40 expression later becomes restricted to the atria andperipheral conduction system (Miquerol et al., “Gap Junctional Connexinsin the Developing Mouse Cardiac Conduction System,” Novartis Found Symp.250:80-98; discussion 98-109, 276-109 (2003), which is herebyincorporated by reference in its entirety), it is plausible that NRG-1βtreatment induced their cells into the latter phenotype.

Studies are underway to determine the source of the endogenous NRG-1βagonist in the hESC-CM cultures described herein. In the developingmouse heart, NRG-1β is known to be released by the endocardium (Meyer etal., “Multiple Essential Functions of Neuregulin in Development,” Nature378(6555):386-390 (1995), Kramer et al., “Neuregulins with an Ig-LikeDomain are Essential for Mouse Myocardial and Neuronal Development,”Proc Natl Acad Sci USA 93(10):4833-4838 (1996), Zhao et al., “SelectiveDisruption of Neuregulin-1 Function in Vertebrate Embryos UsingRibozyme-tRNA Transgenes,” Development 125(10):1899-1907 (1998), andCorfas et al., “Differential Expression of ARIA Isoforms in the RatBrain,” Neuron 14(1):103-115 (1995), which are hereby incorporated byreference in their entirety), but there were vanishingly few endothelialcells in the hESC-CM cultures used in the studies described above.Interestingly, Mercola and colleagues have recently reported that highlypurified hESC-CMs, produced by genetic selection, show less ventricularmaturation than do hESC-CMs in embryoid body preparations of low cardiacpurity (Kita-Matsuo et al., “Lentiviral Vectors and Protocols forCreation of Stable hESC Lines for Fluorescent Tracking and DrugResistance Selection of Cardiomyocytes,” PLoS One 4(4):e5046 (2009),which is hereby incorporated by reference in its entirety). The findingsdescribed herein beg the question whether it is the removal ofNRG-1β-releasing non-cardiac cells that underlies this effect in theirsystem. If so, it may prove helpful to supplement purified hESC-CMs withexogenous NRG-1β when working-type cardiomyocytes are desired.

To conclude, the studies described herein suggest two complementaryapproaches for the generation of subtype-enriched hESC-CMs: 1) geneticlabeling of nodal cells, based on the activation of the proximal cGATA6promoter, and 2) manipulation of NRG-1β/ErbB signaling, which appears toregulate the relative abundance of nodal and working-typecardiomyocytes. Both approaches are expected to have tremendous utilityin cell transplantation studies (e.g., to compare the in vivo behaviorof subtype-enriched preparations), as well as in efforts to furtherelucidate the molecular mechanisms of subtype specification in thedeveloping human heart. It is anticipated that their efficacy in relatedhuman cardiac progenitor cell populations, including cardiomyocytes frominduced pluripotent stem cells (Zhang et al., “Functional CardiomyocytesDerived from Human Induced Pluripotent Stem Cells,” Circ Res.104(4):e30-41 (2009), which is hereby incorporated by reference in itsentirety) and resident cardiac stem cells (Laugwitz et al., “Postnatalisl1+ Cardioblasts Enter Fully Differentiated Cardiomyocyte Lineages,”Nature 433(7026):647-653 (2005) and Messina et al., “Isolation andExpansion of Adult Cardiac Stem Cells from Human and Murine Heart,” CircRes. 95(9):911-921 (2004), which are hereby incorporated by reference intheir entirety) to be similar.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of producing cardiomyocytes having a nodal/pacemakerphenotype, said method comprising: culturing stem cells under conditionseffective to produce cardiomyocytes and contacting the cardiomyocyteswith an antagonist of neuregulin-1 or an antagonist of ErbB underconditions effective to induce production of cardiomyocytes having anodal/pacemaker phenotype.
 2. The method according to claim 1, whereinthe stem cells are selected from the group consisting of embryonic stemcells, adult stem cells, and induced pluripotent stem cells.
 3. Themethod according to claim 1, wherein the stem cells are human embryonicstem cells.
 4. The method according to claim 1, wherein thecardiomyocytes are contacted with an antagonist of neuregulin-1 in theform of an antagonist of neuregulin-1β.
 5. The method according claim 1,wherein the cardiomyocytes are contacted with an antagonist ofneuregulin-1 in the form of a nucleic acid molecule selected from thegroup consisting of a neuregulin-1 antisense molecule, siRNA molecule,and shRNA molecule.
 6. The method according to claim 1, wherein thecardiomyocytes are contacted with an antagonist of neuregulin-1 in theform of an anti-neuregulin-1β antibody or a binding fragment thereof, ora neuregulin-1 aptamer.
 7. The method according to claim 1, wherein thecardiomyocytes are contacted with an antagonist of ErbB in the form of anucleic acid molecule selected from the group consisting of an ErbBantisense molecule, siRNA molecule, and shRNA molecule.
 8. The methodaccording to claim 1, wherein the cardiomyocytes are contacted with anantagonist of ErbB in the form of an anti-ErbB antibody or antibodybinding fragment thereof, or an ErbB aptamer
 9. The method according toclaim 1, wherein the cardiomyocytes are contacted with an antagonist ofErbB in the form of a recombinant protein or peptide fragment thereofselected from the group consisting of Nrdp1, Kek1, argos, and herstatin.10. The method according to claim 1, wherein the cardiomyocytes arecontacted with an antagonist of ErbB in the form of a small moleculeselected from the group consisting of JNJ-28871063, CI-1033, PKI1-66,tryphostin AG825, AG1478, and PD158780.
 11. An isolated population ofnodal/pacemaker cardiomyocytes having a spontaneous firing rate of >90bpm and a slow, biphasic action potential upstroke of <15V/s.
 12. Theisolated population of nodal/pacemaker cardiomyocytes of claim 11,wherein the cardiomyocytes are characterized by a higher level of HCN4expression and a lower level of KCNJ2, SCN5A, and MLC2v expressioncompared to non-nodal/pacemaker cardiomyocytes.
 13. The isolatedpopulation of nodal pacemaker cardiomyocytes of claim 11, wherein thecardiomyocytes are human cardiomyocytes
 14. A pharmaceutical compositioncomprising: the isolated population of nodal/pacemaker cardiomyocytes ofclaim 11; and a pharmaceutically acceptable carrier.
 15. A method oftreating cardiac arrhythmia in a subject, said method comprising:providing the isolated population of nodal/pacemaker cardiomyocytes ofclaim 11 and delivering the isolated population of nodal/pacemakercardiomyocytes to the subject under conditions effective to treat thecardiac arrhythmia.
 16. The method according to claim 15 furthercomprising: selecting a subject having a cardiac arrhythmia, wherein theisolated population of nodal/pacemaker cardiomyocytes is delivered tothe selected subject.
 17. The method according to claim 15, wherein thecardiac arrhythmia is selected from the group consisting of sinus nodedysfunction, bifascicular block, trifascicular block, third-degreeatrial-ventricular block, Stokes-Adam attack, and atrial fibrillation.18. The method according to claim 15, wherein said providing comprises:culturing a population of stem cells under conditions effective toproduce cardiomyocytes and contacting the cardiomyocytes with anantagonist of neuregulin-1 or an antagonist of ErbB under conditionseffective to induce production of cardiomyocytes having anodal/pacemaker phenotype, thereby providing an isolated population ofnodal/pacemaker cardiomyocytes.
 19. The method according to claim 18,wherein the population of stem cells is selected from the groupconsisting of embryonic stem cells, adult stem cells, and inducedpluripotent stem cells.
 20. The method according to claim 18, whereinthe population of stem cells is human embryonic stem cells.
 21. Themethod according to claim 18, wherein the cardiomyocytes are contactedwith an antagonist of neuregulin-1 in the form of an antagonist ofneuregulin-1β.
 22. The method according claim 18, wherein thecardiomyocytes are contacted with an antagonist of neuregulin-1 in theform of a nucleic acid molecule selected from the group consisting of aneuregulin-1 antisense molecule, siRNA molecule, and shRNA molecule. 23.The method according to claim 18, wherein the cardiomyocytes arecontacted with an antagonist of neuregulin-1 in the form of ananti-neuregulin-1 antibody or a binding fragment thereof, or aneuregulin-1 aptamer.
 24. The method according to claim 18, wherein thecardiomyocytes are contacted with an antagonist of ErbB in the form of anucleic acid molecule selected from the group consisting of an ErbBantisense molecule, siRNA molecule, and shRNA molecule.
 25. The methodaccording to claim 18, wherein the cardiomyocytes are contacted with anantagonist of ErbB in the form of an anti-ErbB antibody or a bindingfragment thereof, or an ErbB aptamer.
 26. The method according to claim18, wherein the cardiomyocytes are contacted with an antagonist of ErbBin the form of a recombinant protein or peptide thereof selected fromthe group consisting of Nrdp1, Kek1, argos, and herstatin.
 27. Themethod according to claim 18, wherein the cardiomyocytes are contactedwith an antagonist of ErbB in the form of a small molecule selected fromthe group consisting of JNJ-28871063, CI-1033, PKI-166, tryphostinAG825, AG1478, and PD158780.
 28. The method according to claim 15,wherein said delivering comprises a catheter-based or directintramyocardial injection.
 29. The method according to claim 15, whereinsaid delivering comprises intracoronary infusion.
 30. A method ofproducing cardiomyocytes having an atrial/ventricular phenotype, saidmethod comprising: culturing stem cells under conditions effective toproduce cardiomyocytes; and contacting the cardiomyocytes with aneuregulin-1 agonist, neuregulin mimetic, or an ErbB receptor agonistunder conditions effective to induce the production of cardiomyocyteshaving an atrial/ventricular phenotype.
 31. The method according toclaim 30, wherein the stem cells are selected from the group consistingof embryonic stem cells, adult stem cells, and induced pluripotent stemcells.
 32. The method according to claim 30, wherein the stem cells arehuman embryonic stem cells.
 33. The method according to claim 30,wherein the cardiomyocytes are contacted with an agonist of neuregulin-1in the form or an agonist of neuregulin-1β.
 34. The method accordingclaim 30, wherein the cardiomyocytes are contacted with an agonist ofneuregulin-1 in the form of a recombinant neuregulin-1 protein orpeptide fragment thereof.
 35. The method according to claim 30, whereinthe cardiomyocytes are contacted with an agonist of neuregulin-1 in theform of a nucleic acid molecule encoding a neuregulin-1 protein or apeptide fragment thereof.
 36. The method according to claim 30, whereinthe cardiomyocytes are contacted with an agonist of ErbB in the form ofa recombinant ErbB receptor ligand or ligand fragment.
 37. The methodaccording to claim 30, wherein the cardiomyocytes are contacted with anagonist of ErbB in the form of an ErbB receptor agonist antibody.
 38. Anisolated population of atrial/ventricular cardiomyocytes.
 39. Theisolated population of atrial/ventricular cardiomyocytes of claim 38,wherein the cardiomyocytes have an average spontaneous firing rate of 85bpm and a rapid action potential upstroke of >15 V/s.
 40. The isolatedpopulation of atrial/ventricular cardiomyocytes of claim 38, wherein thecardiomyocytes are characterized by a higher level of SCN5A and KCNJ2expression and a lower level of HCN expression compared tonon-atrial/ventricular cardiomyocytes.
 41. The isolated population ofatrial/ventricular cardiomyocytes of claim 38, wherein thecardiomyocytes are human cardiomyocytes
 42. A pharmaceutical compositioncomprising: the isolated population of atrial/ventricular cardiomyocytesaccording to claim 38; and a pharmaceutically acceptable carrier.
 43. Amethod of improving cardiac tissue repair or cardiac organ function in asubject, said method comprising: providing the isolated population ofatrial/ventricular cardiomyocytes of claim 38 and delivering thepopulation of atrial/ventricular cardiomyocytes to the subject underconditions effective to improve cardiac tissue repair or cardiac organfunction.
 44. The method according to claim 43 further comprising:selecting a subject in need of cardiac tissue repair or improved cardiacorgan function, wherein the isolated population of atrial/ventricularcardiomyocytes is delivered to the selected subject.
 45. The methodaccording to claim 43, wherein said providing comprises: culturing apopulation of stem cells under conditions effective to producecardiomyocytes; and contacting the cardiomyocytes with a neuregulin-1agonist, a neuregulin-1 mimetic, or an ErbB receptor agonist underconditions effective to induce the production of cardiomyocytes havingan atrial/ventricular phenotype, thereby providing an isolatedpopulation of atrial/ventricular cardiomyocytes.
 46. The methodaccording to claim 45, wherein the population of stem cells is selectedfrom the group consisting of embryonic stem cells, adult stem cells, andinduced pluripotent stem cells.
 47. The method according to claim 45,wherein the population of stem cells is human embryonic stem cells. 48.The method according to claim 45, wherein the cardiomyocytes arecontacted with an agonist of neuregulin-1 in the form of an agonist ofneuregulin-1β
 49. The method according claim 45, wherein thecardiomyocytes are contacted with an agonist of neuregulin-1 in the formof a recombinant neuregulin-1 protein or peptide fragment thereof. 50.The method according to claim 45, wherein the cardiomyocytes arecontacted with an agonist of neuregulin-1 in the form of a nucleic acidmolecule encoding a neuregulin-1 protein or a peptide fragment thereof.51. The method according to claim 45, wherein the cardiomyocytes arecontacted with an ErbB receptor agonist in the form of a recombinantErbB receptor ligand or ligand fragment.
 52. The method according toclaim 45, wherein the cardiomyocytes are contacted with an ErbB receptoragonist in the form of an ErbB receptor agonist antibody.
 53. The methodaccording to claim 43, wherein said delivering comprises acatheter-based or direct intramyocardial injection.
 54. The methodaccording to claim 43, wherein said delivering comprises intracoronaryinfusion.